Study of Population Dynamics of Cockroaches Collected from Lahore with Resistance Patterns of Their Isolated Microbial Fauna” Submitted by Ms

STUDY OF POPULATION DYNAMICS OF

COCKROACHES COLLECTED FROM LAHORE

WITH RESISTANCE PATTERNS OF THEIR

ISOLATED MICROBIAL FAUNA

______

HAFSA MEMONA

______

DEPARTMENT OF ZOOLOGY

LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE

2016 STUDY OF POPULATION DYNAMICS OF COCKROACHES

COLLECTED FROM LAHORE WITH RESISTANCE

PATTERNS OF THEIR ISOLATED MICROBIAL FAUNA

______

A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN

PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN ZOOLOGY

HAFSA MEMONA

______

DEPARTMENT OF ZOOLOGY

LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE

2016 CERTIFICATE

This is to certify that the research work described in thesis entitled “Study of population dynamics of cockroaches collected from lahore with resistance patterns of their isolated microbial fauna” submitted by Ms. HAFSA MEMONA to Department of ZOOLOGY, Lahore College for Women University has been carried out under our direct supervision. We have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. We further certify that thesis data have not been used in part or full, in a manuscript already submitted or in the process of submission in partial fulfillment of the award of any other degree from any other institution or home or abroad. We also certify that the enclosed manuscript has been prepared under my supervision and we endorse its evaluation for the award of PhD degree through the official procedure of University.

______Supervisor Co-supervisor Prof. Dr. Farkhanda Manzoor Prof. Dr. Aftab Ahmad Anjum Head Department of Zoology Chairman Department of Microbiology LCWU, Lahore. UVAS, Lahore. Date:

Verified By Chairman

______Prof. Dr. Farkhanda Manzoor Head Department of Zoology Stamp:

______Controller of Examination Stamp Date: ______

DEDICATION

I DEDICATE ALL MY SUCCESS TO MY EVER LOVING PARENTS,

PROF. AHMAD YAR KHAN AND MRS. AZRA PARVEEN

AND MY KIND HEARTED GRANDMOTHER,

MRS. SARDAR BEGHUM (LATE)

WHOSE INSPIRATION, ENCOURAGEMENT, SACRIFICE, MORAL ENTHUSIASM,

FINANCIAL SUPPORT, GUIDANCE, EXPERIENCE, WISDOM

AND KNOWLEDGE MADE THIS STUDY POSSIBLE.

OTHERWISE, WITHOUT THEIR HELP

IT WOULD HAVE BEEN A DREAM FOR ME ACKNOWLEDGMENTS

The whole praise to ALLAH Almighty the sovereign Power, the creator of the universe, who gave us knowledge, with which we are trying to understand the laws of nature and conquer the universe; who made me the super creature; blessed me with knowledge and enabled me to accomplish this task. I offer my humblest and sincere words of thanks to the Holy Prophet

Muhammad (PBUH), who is forever a torch of guidance for humanity.

A special acknowledgement goes to Higher Education Commission, Pakistan for financial support in this fully funded research by awarding me Indigenous scholarship. I feel all words on the earth just failing to express my deep sense of gratitude to our respected Vice

Chancelor, Prof. Dr. Uzma Qureshi for giving me opportunities to complete my research thesis of Ph.D Zoology. I respectfully express my deepest appreciation to our respected, distinguished and affectionate director research Prof. Dr. Shagufta Naz for providing opportunities to complete my research thesis.

I am also obliged to my supervisor Prof. Dr. Farkhanda Manzooor, Head of Zoology

Department for her sincerest guidance, critical suggestions. She has been so kind and supportive with her patience and knowledge and a source of inspiration for me. I respectfully express my deepest appreciation and acknowledgement to my co-supervisor Prof. Dr. Aftab

Ahmad Anjum, Chairman of Microbiology Department, UVAS, Lahore for his keen interest, providing research facilities and valuable suggestions. I am also greatly indebted to my friends, lab fellows for their friendly attitude, support and sympathetic encouragements.

Special thanks to the incharge, Urban Pest Management (UPM) and their team for helping me in sample collection. I also want to pay thanks to laboratory staff of Zoology Department of

LCWU for their cooperation.

HAFSA MEMONA CONTENTS

Title Page no.

List of Tables i

List of Figures iii

List of Abbreviations vi

Abstract xi

Chapter 1 : Introduction 1

Chapter 2: Literature Review 15

Chapter 3: Materials and Methods 44

3.1: Experimental sites 44

3.2: Study area 44

3.3: Collection and identification of cockroaches 44

3.3.1: Collection of cockroaches 44

3.3.2: Identification of cockroaches 47

3.3.3: Weather data collection 47

3.3.4: Data analysis 47

3.4: Isolation and identification of microbes from external surfaces 49 of cockroaches 3.4.1: Sample collection 49

3.4.2: Isolation of bacteria from external surfaces of 49 cockroaches 3.4.3: Identification of bacteria from external surfaces of 49 cockroaches 3.4.3.1: Preparation of media 49

3.4.3.1.1: Preparation of Tryptic Soy Agar 49 (TSA) 3.4.3.1.2: Preparation of Mannitol Salt Agar 49 (MSA) 3.4.3.1.3: Preparation of MacConkey Agar 50

3.4.3.1.4: Preparation of Eosin Methylene 50 Blue Agar (EMB) 3.4.3.1.5: Preparation of Salmonella shigella 50 Agar (SS agar) 3.4.3.1.6: Preparation of Blood Agar Plate 50 (BAP) 3.4.4: Inoculation of sample 51

3.4.5: Identification of microbes 51

3.4.5.1: Microscopic colonial morphology 51

3.4.5.2: Gram staining 51

3.4.5.3: Biochemical tests 52

3.4.5.3.1: Oxidase test 52

3.4.5.3.2: Catalase test 52

3.4.5.3.3: Coagulase test 53

3.4.5.3.3.1: Slide coagulase test 53

3.4.5.3.3.2: Tube coagulase test 53

3.4.5.3.3: Indole test 53

3.4.5.3.4: Methyle Red -Voges Proskaeur 54 (MRVP) test 3.4.5.3.4.1: Methyl red test 54

3.4.5.3.4.2: Voges-Proskauer (VP) 54 test 3.4.5.3.5: Citrate test 55

3.4.6: Statistical analysis of bacterial isolates 55

3.5: Isolation and identification of bacteria from alimentary tract of 57 cockroaches 3.5.1: Collection of samples 57

3.5.2: Isolation of bacteria from alimentary tract of 57 cockroaches 3.5.3: Identification of microbes from alimentary tract of 57 cockroaches 3.5.3.1: Preparation of media 57

3.5.3.2: Inoculation of sample 57

3.5.3.3: Identification of microbes 57

3.5.4: Statistical analysis of microbial isolates from 58 alimentary tract of cockroaches 3.6: Isolation of fungal flora from cockroaches 59

3.6.1: Preparation of samples 59

3.6.2: Preparation of media for fungal culturing 59

3.6.2.1: Preparation of Sabouraud’s Dextrose Agar 59 (SDA) 3.6.2.2: Preparation of Malt Extract Agar (MEA) 60

3.6.3: Inoculation of samples 60

3.6.4: Identification of fungal isolates 61

3.7: Isolation of parasitic contaminants from external surfaces of 61 cockroaches 3.7.1: Preparation of samples 61

3.7.2: Identification of parasitic contaminants 61

3.7.3: Statistical analysis 62

3.8: Evaluation of antimicrobial sensitivity 62

3.8.1: Preparation of Tryptic Soy Broth (TSB) 63

3.8.2: Inoculation of sample in Tryptic Soy Broth (TSB) 63

3.8.3: Preparation of Mueller Hinton Agar plates 63

3.8.4: Inoculation of samples on Muller Hinton Agar 64

3.8.5: Analysis of results 64

3.9: Efficacy of common disinfectants for bacterial isolates 64

3.9.1: Preparation of MH Agar plates 65

3.9.2: Inoculation of samples 65

3.9.3: Serial dilution of disinfectants 65 3.9.4: Well diffusion method 65

3.10: Quantitative and qualitative analysis of total protein extracted 66 from resistant bacterial isolates 3.10.1: Preparation of reagents 67

3.10.1.1: 1X Phosphate Buffer Saline (PBS) 67

3.10.1.2: 1X cell lysis buffer 67

3.10.1.3: Phenylmethylsulfonyl fluoride (PMSF) 67 solution 3.10.2: Protein extraction procedure 67

3.10.3: Bradford protein assay 67

3.10.3.1: Preparation of Bradford reagent 68

3.10.3.2: Preparation of dilutions of standard protein 68 sample {Bovine Serum Albumin (BSA)} 3.10.3.3: Macro assay 68

3.10.3.4: Calculations for unknown samples 68

3.10.4: SDS-Polyacrylamide Gel Electrophoresis (SDS- 69 PAGE) 3.10.4.1: Reagent preparation 69

3.10.4.1.1: 30% Acrylamide/ Bisacrylamide 69 solution 3.10.4.1.2: Tris-HCl 1.5 M (pH 8.8) 69 Resolving gel buffer 3.10.4.1.3: 1 M Tris-HCl (pH 6.8) Stacking 69 gel buffer 3.10.4.1.4: 10% Sodium Dodecyl Sulphate 69 (SDS) Solution 3.10.4.1.5: 10% Ammonium per Sulphate 70 (APS) solution 3.10.4.1.6: 1X Running buffer (SDS 70 electrophoresis buffer) 3.10.4.1.7: Tracking dye (loading dye) 70

3.10.4.1.8: Coomassie stain (staining 70 solution) 3.10.4.1.9: Coomassie destain (destaining 70 solution) 3.10.4.1.10: Preparation of working dilutions 70

3.10.4.1.11: Protein marker 71

3.10.4.2: Gel preparation 71

3.10.4.2.1: 10% Resolving gel preparation 71

3.10.4.2.2: 5% Stacking gel preparation 71

3.10.4.3: Gel electrophoresis 71

3.10.4.4: Staining 72

3.10.4.5: Destaining 72

3.10.4.6: Image capture and photography 72

3.10.4.7: Quantification of protein fractions 72

Chapter 4: Results 73

4.1: Collection and Identification of Cockroaches 73

4.1.1: Identified species of cockroaches 73

4.1.2: Distribution and abundance of cockroach species 76

4.1.2.1: First Trimester 76

4.1.2.2: Second Trimester 76

4.1.2.3: Third Trimester 77

4.1.2.4: Fourth Trimester 77

4.1.2.5: Relative abundance 79

4.2: Isolation of microorganisms from external and internal surfaces 83 of cockroaches 4.2.1: Identified bacterial species 83

4.2.2: Bacterial infection rate at hospitals and houses 88

4.2.3: Ecological indices of isolated microorganisms 90

4.3: Isolation of fungal flora from cockroaches 91

4.3.1: Variety of fungal isolates 91 4.3.2: Prevalence of fungal flora isolated from P. americana 98 and B. germanica 4.4: Isolation of parasitic contaminants from cockroaches 99

4.4.1: Identified species of parasites isolated from 99 cockroaches 4.4.2: Ecological indices of parasitic contaminants of 102 cockroaches 4.5: Evaluation of antimicrobial sensitivity 107

4.6: Efficacy of common disinfectants for bacterial isolates 110

4.7: Quantitative and qualitative analysis of total protein extracted 114 from resistant bacterial isolates 4.7.1: Bradford Micro Assay 114

4.7.2: SDS-PAGE for total protein electrophoresis 115

Chapter 5: Discussion 122

References 138

Annexures xiii

Plagiarism Report xxv

List of Publications and reprints xxvii i

List of Tables

Table no. Title Page no. 3.3.1: GPS location of cockroach collection sites in urban area of Lahore, 45 Pakistan. 3.8: List and class of antibiotics used in the present study. 63 4.1.2a: Average minimum and maximum air temperatures and relative humidity 77 (%) recorded from April 2013 to March 2014 (four trimesters). 4.1.2b: Diversity indices of different cockroach species collected during four 78 trimesters of sampling (April 2013– March 2014). 4.1.2c: Sampling location types and number of cockroaches belonging to 80 different species trapped from the various experimental sites. 4.1.2d: Air temperature, humidity, mean, standard deviation and one-way 81 ANOVA values of different cockroach species collected in different trimesters from urban locations in Lahore, Pakistan. 4.1.2e: Coefficient of correlation between environmental factor (air temperature 82 and relative humidity) and populations of different cockroach species collected from urban areas of Lahore, Pakistan. 4.1.2f: Post hoc comparisons using the Fisher LSD test for the one-way 82 analysis of variance between air temperature and populations of different cockroach species collected in Lahore, Pakistan. 4.2.1a: Details of identification and biochemical characters used for classical 87 identification of bacterial isolates from cockroaches collected in Lahore, Pakistan. 4.2.1b: Relative abundance of bacterial isolates harbored on external and 88 internal body surfaces of cockroaches (n = 110) collected in Lahore, Pakistan. 4.2.2a: External and internal bacterial infection rate of hospital cockroaches in 89 different hospitals of Lahore, Pakistan. 4.2.2b: External and internal bacterial infection rate of cockroaches collected 89 from different houses of Lahore, Pakistan. 4.2.3: Community diversity indices of bacterial species in and on cockroaches 90 collected from different habitats (hospitals and houses) in Lahore, Pakistan. 4.3.1: Prevalence of fungal contaminants isolated from cockroaches (n=60) 92 and their identification characteristics. 4.3.2: Prevalence of fungal contaminants in cockroaches collected from 98 various houses of Lahore, Pakistan. 4.4.2a: Prevalence and ecological indices of parasitic contaminants isolated 103 from P. americana and B. germanica collected from houses of Lahore, Pakistan.

4.4.2b: Prevalence and ecological indices of parasitic contaminants isolated 104 from P. americana and B. germanica collected from hospitals of Lahore. 4.4.2c: Mean, standard deviation, one-way ANOVA of parasites isolated from 106 cockroaches collected from houses and hospitals of urban area of Lahore, Pakistan. 4.5a: Antibiotic sensitivity profile of bacterial isolates obtained from 109 cockroaches (P. americana and B. germanica) 4.5b: Antibiotic resistance profile of bacterial isolates obtained from 109 cockroaches (P. americana and B. germanica) 4.6a: Means + SE values of inhibition zone diameter using 3 different 111 concentrations of 3 different disinfectants against bacteria isolated from cockroaches. 4.6b: Effectiveness of three disinfectant agents on bacteria isolated from 113 cockroaches. 4.7.1: Absorbance and concentration of the standard (BSA) and the samples at 114 595 nm. 4.7.2a: Lane-wise molecular weight description of Bands of 10% SDS-PAGE 120 gel 1. 4.7.2b: Lane-wise molecular weight description of Bands of 10% SDS-PAGE 121 gel 2.

iii

List of Figures

Figure no. Title Page no. 3.3.1: Map of Lahore District showing the collection site of cockroaches 46 including hospitals and houses. 3.10.3.3: Standard curve for Bradford assay. 68 4.1.1a: Pictorial presentation of American cockroach {P. americana (A: 74 female, B: male)} collected from Lahore, Pakistan. 4.1.1b: Pictorial presentation of German cockroach {B. germanica (A: 74 male, B: female laying ootheca, C: nymph)} collected from Lahore, Pakistan. 4.1.1c: Pictorial presentation of Oriental cockroach {B. orientalis (A: 74 female, B: male laying ootheca)} collected from Lahore, Pakistan. 4.1.1d: Pictorial presentation of Turkestan cockroach {B. lateralis (A: 74 male, B: female)} collected from Lahore, Pakistan. 4.1e-g: Cockroach infested sites in different houses of Lahore, Pakistan. 75 4.2.1a: Growth of E. coli on EMB agar plates. 84 4.2.1b: Growth of E. coli on MacConkey agar plates. 84 4.2.1c: K. pneumoniae pure culture growth on MacConkey (left) and EMB 84 agar (right) plates. 4.2.1d: Mannitol Salt agar plates showing pure growth lawn of S. 85 pneumonia. 4.2.1e: Mannitol Salt agar plates showing pure growth lawn of S. aureus. 85 4.2.1f: EMB agar plate showing pure growth of P. aeruginosa. 85 4.2.1g: Salmonella Shigella agar plate showing growth of S. dysentriae. 85 4.2.1h: Mannitol salt agar plate showing growth of S. pyogenes and S. 86 epidermidis. 4.2.1i: Nutrient agar plate showing growth of S. aureus. 86 4.2.1j: Biochemical test results of Citrate test. 86 4.2.1k: Biochemical test results of Catalase test. 86 4.3.1a: Geotrichum candidum colony grown on Malt Extract agar. 93 4.3.1b: A. flavus colony grown on Malt Extract agar plate. 93 4.3.1c: A. alternata colony grown on Malt Extract agar plate. 93 4.3.1d: A. zonatus colony grown on Malt Extract agar plate. 93 4.3.1e: M. anisopoliae colony grown on Malt Extract agar plate. 94 4.3.1f: Mucor spp. grown on Malt Extract agar plate. 94 4.3.1g: Aspergillus spp. grown on Malt Extract agar plate. 94

4.3.1h: A. oryzae colony grown on Malt Extract agar plate. 94 4.3.1i: Micrograph of conidia of A. flavus isolated from external surfaces 95 of cockroaches. 4.3.1j: Micrograph of conidia of A. oryzae isolated from external surfaces 95 of cockroaches. 4.3.1k: Micrograph of conidia of G. candidum isolated from external 95 surfaces of cockroaches. 4.3.1l: Micrograph of conidiophore of A. flavus isolated from external 96 surfaces of cockroaches. 4.3.1m Micrograph of conidiophore of A. oryzae isolated from external 96 surfaces of cockroaches. 4.3.1n Micrograph of conidiophore of Metarhizium spp. isolated from 96 external surfaces of cockroaches.

4.3.1o Micrograph of conidiophore of G. candidum isolated from external 97 surfaces of cockroaches. 4.3.1p Micrograph of conidiophore of A. zonatus isolated from external 97 surfaces of cockroaches. 4.4.1a: A. lumbricoides eggs isolated from body surface of cockroaches. 100 4.4.1b: E. vermicularis eggs isolated from body surface of cockroaches. 100 4.4.1c: E. vermicularis larvae isolated from body surface of cockroaches. 100

4.4.1d: Larvae of S. stercoralis isolated from body surface of cockroaches. 100 4.4.1e: Ova of T. trichura isolated from body surface of cockroaches. 101 4.4.1f: Cysts of E. histolytica isolated from body surface of cockroaches. 101 4.4.1g: Oocyst of C. parvum isolated from body surface of cockroaches. 101 4.4.1h: Oocyst of I. belli isolated from body surface of cockroaches. 101 4.4.2a: Prevalence of parasites isolated from the P. americana and B. 105 germanica collected from houses in Lahore, Pakistan. 4.4.2b: Prevalence of parasites isolated from the P. americana and B. 105 germanica collected from hospitals in Lahore, Pakistan. 4.5a: Antimicrobial sensitivity pattern of S. aureus. 108 4.5b: Antimicrobial sensitivity pattern of E. coli. 108 4.5c: Antimicrobial sensitivity pattern of S. typhi. 108 4.5d: Antimicrobial sensitivity pattern of P. aeruginosa. 108 4.6a: Zones of inhibition of disinfectants used against S. aureus in well 112 diffusion method. 4.6b: Zones of inhibition of disinfectants used against E. coli in well 112 diffusion method.

4.7.2a: 10% SDS-PAGE gel 1 representing crude protein band patterns of 116 resistant bacterial isolates of cockroaches. 4.7.2b: Lane-wise individual band molecular weight representation of 117 SDS-PAGE gel 1. 4.7.2c: 10% SDS-PAGE gel 2 representing crude protein band patterns of 118 resistant bacterial isolates of cockroaches. 4.7.2d: Lane-wise individual band molecular weight representation of 119 SDS-PAGE gel 2.

List of Abbreviations

A. alternate Alternaria alternate A. anitratus Acinetobacter anitratus ABU Ahmadu Bello University A. baumannii Acinetobacter baumannii A. diaperinus Alphitobius diaperinus AML Amoxicillin A. duodenalae Ancylostoma duodenaale ANOVA Analysis of variance A. fumigatus Aspergillus fumigatus A. flavus Aspergillus flavus AIIMS All India Institute of Medical Sciences A. lumbricoides Ascaris lumbricoides A. niger Aspergillus niger A. oryzae Aspergillus oryzae API Analytical profile index APS Ammonium per sulphate A. viridans Aerococcus viridans A. zonatus Aspergillus zonatus B. asahinai Blattela asahinai (Mizukubo) B. cereus Bacillus cereus B. coli Balantidium coli B. germanica Blattella germanica B. hominis Blastocystis hominis B. lateralis Blattella lateralis B. lituricollis Blattella lituricollis (Walker) B. orientalis Blatta orientalis B. vaga Blattella vaga (Hebard) BAP Blood agar plates C. albicans Candida albicans C. brakii Cutrobacter brakii C. cayetanensis Cyclospora cayetanensis C. celluovorans Clostridium cellulovorans C. difficile Clostridium difficile

vii

CDC Center for Disease control and Prevention CE Cephradine CFU Colony forming unit C. freundii Citrobacter freundii C. glabrata Candida glabrata C. guilliermondii Candida guilliermondii CIP Ciprofloxacin CLSI Clinical and Laboratory Standards Institute C. macellaria Cochliomyia macellaria C. mansilli Chilomastix mesnilli CNS coagulase negative staphylococci C. parvum Cryptosporidium parvum C. parapsilosis Candida parapsilosis CRO Ceftriaxone C. tropicalis Candida tropicalis D. melanogaster Drosophila melanogaster D. punctate Diploptera punctata DTT Dithiothreitol E. aerogenes Enterobacter aerogenes E. blatti Entamoeba blatti E. cloacae Enterobacter cloacae E. coli Escherichia coli E. coli Entameba coli E. faecalis Enterococus faecalis E. faecium Enterococcus faecium E. histolytica Entameba histolytica E. nana Endolimax nana EMB Eosine methylene blue agar EPEC Enteropathogenic E. coli ESBL Extended spectrum of beta lactamases E. vermicularis Enterobius vermicularis FFCBP First fungal culture bank of Pakistan F. moniliformes Fusarium moniliformei G. candidum Geotrichum candidum G. duodenalis Giardia duodenalis

viii

G. robustus Gordius robustus GS-MRSA Gentamycin Sensitive MRSA GPS Global Positioning System HAIs Hospital Associated Infections HIV Human Immunodeficiency Virus H. concinna Hebardina concinna (Dehaan) H. diesingi Hammersmiditiella diesingi H. influenza Haemophilus infuenzae I. belli Isospora belli I. butschlii Iodamoeba butschlii J. erebis Jacobsonina erebis K. oxytoca Klebsiella oxytoca K. pneumonia Klebsiella pneumoniae L. monocytogenes Listeria monocytogenes LSD Least significant difference MAR Multiple antibiotic resistance MBC Minimum Bactericidal Concentration MDR Multi drug resistant M. domestica Musca domestica M. dubius Moniliformis dubius MEA Malt extract agar MH agar Mueller Hinton agar MIC Minimum Inhibitory Concentration MICU medical ICU MRCNS Methicilin Resistant CNS MRSA Methicilin Resistant Staphylococcus aureus MRVP Methyl red voges proskaeur MSA Mannitol salt agar M. moniliformis Moniliformis moniliformis M. morganii Morganella morganii M. tuberculosis Mycobacterium tuberculosis M. varians Micrococcus varians N. cinerea Nauphoeta cinerea (olivier) N. gonorrhoeae Neisseria gonorrhoeae NICU Neonatal Intensive care unit

NIH National institute of health NNIS National Nosocomial Infection Surviellance N. rhombifolia Neostylopyga rhombifolia (Stool) N. ovalis Nyctotherus ovalis PAGE Polyacrylamide Gel Electrophoresis P. aegyptica Polyphaga aegyptica P. aeruginosa Pseudomonas aeruginosa P. Americana Periplaneta Americana P. australasiae Periplaneta australasiae P. brunnea Periplaneta brunnea PBS Phosphate buffer saline PHMB Poly hexamethylene biguanide P. fuliginosa Periplaneta fuliginosa PIC Punjab Institute of Cardiology P. japonica Periplaneta japonica PMSF Phenylmethylsulfonyl Fluoride P. maltophilia Pseudomonas maltophilia P. mirabilis Proteus mirabilis P. pensylvanica Periplaneta pensylvanica P. saussurei Polyphaga saussurei P. surinamensis Pycnoscelis surinamensis (Linnaeus) P. vulgaris Proteus vulgaris R. madera Rhyparobia (Leucophea) maderae S. aureus Staphylococcus aureus S. anatum Salmonella anatum S. brevicaulis Scopulariopsis brevicaulis S. carnaria Sarcophaga carnaria S. calcitrans Stomoxys calcitrans S. dysentriae Shigella dysenteriae SDA Sabouraud dextrose agar SDS Sodium dodecyl sulphate S. evidens Symploce evidens S. epidermidis Staphylococcus epidermidis S. equorum Staphylococcus equorum S. Flexner Shigella Flexner

S. faecalis Streptococcus faecalis S. haematobium Schistosoma haematobium S. longipalpa Supella longipalpa S. liquefaciens Serratia liquefaciens S. marcescenes Serratia marcescenes S. mansoni Schistosoma mansoni S. miyakoensis Symploce miyakoensis S. okinoerabuensis Symploce okinoerabuensis S. oranienburg Salmonella oranienburg S. pallens Symploce pallens S. paramarginata Symploce paramarginata S. pneumonia Streptococcus pneumoniae S. sphaerica Symploce sphaerica S. stercoralis Strongyloides stercoralis SS agar Salmonella Shigella agar STH soil transmitted helminthes S. typhi Salmonella typhi S. typhosa Salmonella typhosa S. typhimurium Salmonella typhimurium T. beigelii Trichosporon beigelii T. bulhoe Thelastoma bulhoe T. trichiura Trichuris trichiura TBC Total Bacterial count TE Tetracycline TEMED N-N-N’-N’-Tetramethylethylenediamine TSA Tryptic Soy agar TSB Tryptic Soy Broth VRE Vancomycin-resistant enterococci V. cholera Vibrio cholera

ABSTRACT

Cockroaches are one of the most important pests in urban communities and are risky for human health because they play an important role in transmitting different diseases either mechanically or occasionally biologically. Environmental and sanitary conditions associated with demographic and socio-economic settings of an area could contribute to the prevalence of disease pathogens carried by cockroaches. The present study was aimed to determine taxonomical identification and diversity of cockroaches in hospitals and houses in Lahore, Pakistan and to evaluate the role of cockroaches in transmission of important disease pathogens by using microbial screening of outer surface and digestive tract. Resistance and susceptibility to antimicrobials and disinfectants was also investigated, followed by quantitative and qualitative analysis of total bacterial protein. Four species of cockroaches (P. americana, B. germanica, B. orientalis and B. lateralis) were identified in this study. B. germanica was found to be the most dominant indoors species with highest diversity indices followed by P. americana. Species diversity was highest in July–September, 2013 with highest Simpson index of diversity and Shannon index as well. Population index of B. germanica for hospitals was double than that of residential areas. Houses and hospitals were highly infested with P. americana and B. germanica as compared to offices, shopping malls/ departmental stores and universities. Whereas B. orientalis was commonly found in houses, institutes/universities followed by hospitals, while B. lateralis was common in institutes/ universities, houses and offices with basements and gardens. P. americana was found higher in all trimesters (mean 1179.50 + 351.77) and the analysis of variance revealed a significant difference between the groups (F(3,4)=9.65, P=0.005). Significant correlation was found among changes in temperature and abundance of P. americana in study area (Pearson correlation, r= 0.904, P= 0.04). Similarly temperature showed positive correlation with population of B. germanica (r= 0.958, P= 0.021) and B. orientalis (r= 0.987, P= 0.007).

During the study, all cockroaches were found to be infected with at least one bacterium. The most common bacterium isolated from external surface of cockroaches was E. coli (10.31%), followed by S. aureus (10.09%), while P. aeruginosa (19.96%) was isolated from internal gut tract of cockroaches, followed by P. vulgaris (16.08%). Among hospitals the highest in external bacterial infection was observed on P. americana in Punjab Institute of Cardiology (PIC) (75.6%) while

xii highest internal bacterial infection was observed in Sheikh Zaid Hospital (SZH) (30.8%). Same trend was observed for bacterial isolation from B. germanica in hospitals. Among houses highest external bacterial infection for P. americana (55.9%) and B. germanica (52%) was observed in samples collected from Shalamar- II. However, highest internal contamination (25.8%) for P. americana was observed in Mughalpura-I locality while highest internal infection (28.8%) for B. germanica was Model town-3 houses. Jaccard’s index of similarity was highest (0.3125) in houses while Bray-Curtis index of dissimilarity was highest for hospital (0.2174). The highest Shannon-Wiener’s diversity index value was found for Punjab Institute of Cardiology (2.610632). All cockroaches had also carried one or more species of medically important mould on their external surface.

In this study the most common mold isolated and identified from First Fungal Culture Bank of Pakistan were A. oryzae (84%) and A. flavus (75%) while least common was G. candidum (22%). Cockroaches collected from Shadman-1, Johar town-1 and Shalamar-2 showed relatively high fungal prevalence. P. americana harboured more parasites as compared to B. germanica in both environment. E. coli protozoan was found as the most prevalent followed by E. vermiculari however, A, lumbricoides were least prevalent in hospitals and houses. Simpson Diversity index value of parasitic contaminants isolated from B. germanica collected from houses was 0.92133 and 0.91827 for hospitals. The Shannon-Weiner diversity index calculated value was found highest for P. americana at both sites houses and hospitals as 2.554291 and 2.536765 respectively, which predicted that the rate of parasitic contaminants of both species was not even. Both experimental sites were not significantly different in carriage of parasitic contaminants on cockroaches (F (1,6) =1.795, P= 0.229). Resistance to amoxicillin was found 100% for both gram negative and gram positive isolates followed by cephradine and tetracycline respectively. E. coli was observed as resistant to 3 out of 5 antibiotics (AML, CE, TE) followed by P. aeruginosa that showed resistance to amoxicillin and tetracycline. Germ Kill Vantocil FHC was found more effective bactericide than Germ kill Vantocil in current study. Germ kill Vantocil FHC exhibit highest inhibition zone diameter mean (27+11.575) for 12.5% diluton and 14+13.856 at 50% dilution. Similarly RIZD was 81.81% for 12.5%, dilution respectively. Protein bands of control group were compared with resistant bacterial samples and all protein bands are lying between 236216.2 kDa – 10000 kDa.

ABSTRACT

ii highest internal bacterial infection was observed in Sheikh Zaid Hospital (SZH) (30.8%). Same trend was observed for bacterial isolation from B. germanica in hospitals. Among houses highest external bacterial infection for P. americana (55.9%) and B. germanica (52%) was observed in samples collected from Shalamar- II. However, highest internal contamination (25.8%) for P. americana was observed in Mughalpura-I locality while highest internal infection (28.8%) for B. germanica was Model town-3 houses. Jaccard’s index of similarity was highest (0.3125) in houses while Bray-Curtis index of dissimilarity was highest for hospital (0.2174). The highest Shannon-Wiener’s diversity index value was found for Punjab Institute of Cardiology (2.610632). All cockroaches had also carried one or more species of medically important mould on their external surface.

INTRODUCTION

Among the major phyla in the animal kingdom which colonize and establish successfully on land and sea is the phylum Arthropoda. It is the largest phylum in all described species of animals. Included in phylum Arthropoda, 90% are insects and 10% other diverse and prolific subphyla (Triplehorn and Johnson, 2005). In the world insects are the most diversified, abundant and cosmopolitan animals representing more than 50% of all species living on the planet earth, 71% of animal species, 74% of invertebrates, and 87% of all arthropods (Kenawy et al., 2014).

Synanthopic insects living in human habitats get their food from industries, houses, field crops and rejected food dumped in landfills and sewage. These insects are granivorous, parasitic, phytophagous and saprophagous. In urban and rural areas, the great majority of synanthropic insects are present (Mikulak et al., 2013). Non-biting flies, ants and cockroaches are included in synanthropic insects which are efficient mechanical vectors of human-related protozoan parasites, disease-causing microorganisms from the contaminated environment to human food, kitchen crockery and medical tools in hospitals. They carry pathogenic organisms internally or externally on their body and play an important role in spreading the pathogens by carrying and transmitting them ultimately to a definitive and intermediate host (Pesquero et al., 2012).

The most successful and ancient form of insects are cockroaches. Cockroaches had been around since Pennsylvanian times and attained a suitable body form to become successful group on land since that time. Approximately 4500 species of cockroaches have been classified to date but most of the cockroach species are domiciliary pests (Cochran, 1983). They have dorsoventrally flattened body, head covered beneath the pronotum, chewing mouth parts and having prominent antennae and cerci in both sexes (Tsai and Lee, 2001).

All known species of cockroaches which act as pests are less than 1% of their total population. Free-living species of cockroaches mainly exist in tropical regions of the world. They can also live in varied environment such as under stones, dead and decaying leaves, on flowers and grass, in semi-aquatic environments, in the caves and burrows and in the nest of ants, termites and wasps (Cochran, 2001). 2

Cockroaches eat everything available to them but they prefer the food source like fruits, vegetables, sweets and meat. However, they prefer to live in warm environments with abundant food sources; wet decaying areas like kitchens are their natural habitat (Kassiri et al., 2014a). Cockroaches have been commonly observed to occur in varied environment like hospitals, landfill sites and food industries (Gadd and Raubenheimer, 2000; Chaichanawongsaroj et al., 2004; Jalil et al., 2012; Shahraki et al., 2013).

If an organism is able to survive under extreme temperatures (during winter or summer) their likelihood to colonize that habitat will increase. The “heat shock proteins (HSP)” in the cells of insects which allow recovery at cellular level are found in many organisms from bacteria to mammals (Lutterschmidt and Hutchison, 1997).

Organisms have the ability to increase or decrease their core temperature in response to environmental temperatures. Termites have the ability to acclimate to their environmental temperature (Hu and Appel, 2004). Since cockroaches are closely related to termites, they should have the tendency to acclimatize to their environment. Several previous studies have shown a positive correlation between the temperature sensitivity of many animals including cockroaches and their environmental temperature (Tsuji and Mizuno, 1973; Slabber et al., 2007; Appel et al., 2009).

The cockroach species associated with human habitats amount to 30 in number but the species infesting human dwellings are fewer. The most popular species of cockroaches belong to five families occurring in various types of human habitats in China, Kuala Lumpur Federal Territory, Thailand, India, Peninsular Malaysia, Singapore, Bangkok, Indonesia and Pakistan. Family Blattidae include [Periplanata americana L. (P. americana)] (American cockroach), [Periplaneta australasiae (P. australasiae)] (Australian cockroach), [Periplaneta brunnea (P. brunnea)] (Large brown cockroach), [Periplaneta fuliginosa (P. fuliginosa)], [Periplaneta japonica (P. japonica)] (Japanese cockroach), [Blatta orientalis L. (B. orientalis)] (Oriental cokroach), [Neostylopyga rhombifolia (Stool) (N. rhombifolia)] The Harlequin cockroach, [Blatta lateralis (B. (Shelfordella) lateralis] (Walker) (Turkistan cockroach) and Hebardina concinna (Dehaan) (H. concinna) (Walker, 1994; Vythilingam et al., 1997; Cochran, 2001; Sriwichai et al., 2002; Chompoosri et al., 2004; Feizhaddad et al., 2012; Lihoreau et al., 2012). 3

Family Blattelidae include [Blattella germanica L. (B. germanica)] (German cockroach), Blattella lituricollis (Walker (B. lituricollis) (Smaller German cockroach), [Supella longipalpa (S. longipalpa (Fabricius)] (Brown banded cockroach), Blattella vaga (Hebard) (B. vaga) (Field cockroach), Blattela asahinai (Mizukubo) (B. asahinai) (Asian cockroach), Jacobsonina erebis (J. erebis), Symploce pallens (S. pallens) (Smooth cockroach), Symploce sphaerica (S. sphaerica), Symploce miyakoensis (S. miyakoensis), Symploce okinoerabuensis (S. okinoerabuensis), Symploce paramarginata (S. paramarginata) and Symploce evidens (S. evidens). Family polyphagiadae include Polyphaga aegyptica (P. aegyptica) and Polyphaga saussurei (P. saussurei). Family Blaberidae include Rhyparobia (Leucophea) maderae (R. madera) (Madeira cockroach). Family Pycnoscelidae include Pycnoscelis surinamensis (Linnaeus) (P. surinamensis) the Surinam cockroach. Family Oxyhaloidae include Nauphoeta cinerea (olivier) (N. cinerea) the lobster cockroach (Wang and Che, 2013; Jeffery et al., 2012; Boyer and Rivault, 2004(a,b); Cochran, 2001; Zahedi and Jeffery, 1996).

In human habitat, poor sanitation and disrepair increases the cockroach population. Foodstuffs can be contaminated with cockroach faeces, body parts and pathogens. Sometimes, the life of people can be threatened by increase in population of cockroaches inside the homes as they cause asthmatic allergies (Lamiaa et al., 2007). Cockroach faeces are a mixture of xanthourenic acid, cinnamic acid and 8-hydroxy quinaldic acid. They have characteristics of mutagenesis and carcinogensis and are tryptophan derivatives (Cochran, 2001).

Cockroaches have high carriage rate of pathogens of humans as they feed on sewage and garbage. They are ideal carriers for pathogenic organisms because they encounter dirty habitats and have nocturnal foraging habits (Bouamama et al., 2010; Akbari et al., 2015). Cockroaches play an important role in the transmission of nosocomial disease agents from the sewage and landfills to humans because these sites are major sources of pathogenic microorganism (Devi and Murray, 1991; Menasria et al., 2014). Large numbers of cockroaches and houseflies dispersed in unsanitary conditions in urban and rurals areas are responsible for spreading many pathogens that cause bacillary dysentery, typhoid and cholera (Graczyk et al., 2001; Banjo et al., 2005). 4

Presence of cockroaches in kitchen areas point towards the poor food disposal infrastructure (Malik et al., 2013). In humans, cockroaches may be a cause of food poisoning as they also spread fungi, bacteria, viruses, protozoans and intestinal parasitic worm as intermediate hosts (Purcell & Almeida, 2004; Chitsazi et al., 2012; Chitsazi et al., 2013; Solomon et al., 2016).

The common microbiota of insects play an important role in nutrition and food digestion, protection against pathogens, host mating preferences, resistance against parasitoids and detoxification of noxious compounds. Cockroaches present in human environment facing poisonous substances including insecticides, pollutants and other xenobiotics on daily basis. They overcome toxic xenobiotics by their detoxification abilities and oxidative stress. Due to the microbiota in their midgut, they have high digestive ability and adaptation to complex environment (Ma et al., 2009).

The microbiota in the midgut of cockroaches belong mainly to four phyla: Firmicutes, Bacteroidetes, Actinobacteria and proteobacteria. Digestion, detoxification or oxidative stress in insects is caused by orders Clostridiales, Lactobacillales and Burkholderiales, respectively. Lactobacillales potentially play a role in conferring diamondback moth resistance to the insecticides fipronil and chlorpyrifos. Interestingly, Clostridium cellulovorans (C. celluovorans) is the most important species in the P. americana midgut because it contain a cellulosome that digests the cell wall (Sleat et al., 1984; Xia et al., 2013; Zhang et al., 2016).

Over 100 species of bacteria isolated from cockroaches are the dominant vectors that spread infection with the fecal oral route (Prado et al., 2002; Pai et al., 2003; Graczyk et al., 2005). As compared to B. germanica and B. lateralis, P. americana carry more bacterial species in human dwelling localities (Schauer et al., 2012; Prado et al., 2002; Pai et al., 2003). Many years ago it had been reported that cockroaches carry pathogenic bacteria on their external and internal surfaces. Longfellow (1913) was the first who studied about microbial transmission by cockroaches. The commonest pathogens which carried by cockroaches are Proteus vulgaris (P. vulgaris), Staphylococcus aureus (S. aureus) and Bacillus spp.

On external surfaces of cockroaches, many common microorganisms and several potent pathogens occur. The most common microorganisms are Enterobacter spp., Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella 5

pneumonia (K. pneumoniae) (Saitou et al., 2009; Zurek and Schal, 2004). Usually K. pneumonia a gram-negative bacterium is found in the gut of B. germanica. P. aeruginosa can multiply and can be excreted for upto 114 days after ingestion by insects. Salmonella typhi (S. typhi), Shigella dysenteriae (S. dysentriae) and toxigenic strains of E. coli can also be restrained in the gut of cockroaches for several days (Stek, 1982). Cockroaches may cause dangers in the dairy industry by carrying microorganisms including Salmonella, Conidia of mycotoxigenic fungi, Pseudomonas, E. coli and Listeria monocytogenes (L. monocytogenes) (Fakoorziba et al., 2010).

Cockroaches also act as vectors for many infective organisms such as Helminth parasites, cysts of Protozoa, enterovirus and enteropathogenic bacteria. Therefore, they are notorious in veterinary medicines. Other harmful vectors may ride on them by the intake of human enteropathogenic contaminated food (Graczyk et al., 2001).

Indoor infestation of cockroaches is a major cause of mechanically transmitted parasites in hospitals. For mechanical disease transmission, the distinctive eating and living habits of cockroaches mainly mobility and body structure make them a good vector. The level of allergens on cockroaches is directly related to cockroach density, hospital disrepair, and sanitary conditions of that area (Vahabi et al., 2011; Kassiri et al., 2014b).

In hospitals, cockroaches are considered as possible carrier of nosocomial infections, especially the transmission of drug-resistant bacteria (Saitou et al., 2009). Cockroaches also play a vital role in transmitting food-borne diseases, such as typhoid fever, cholera, tuberculosis, dysentery and diarrhea. Cockroaches are important vectors of TEM-type beta-lactamases producing gram-negative bacteria. It has been concluded that the presence of E. coli and K. pneumoniae on cockroaches play role in the epidemiology of nosocomial infections (Graczyk et al., 2005).

The rate of nosocomial infection is much less in industrialized countries as compared to developed countries because they follow standard legislative measures to overcome the disease and death rate. In 2002, the World Health Organization (WHO) surveyed the developing countries and found high rate of nosocomial infections in Eastern Mediterrean and South East Asia. The reasons for this may be the wide use of 6

antibiotics, overpopulation, unhygienic environments which factors may increase resistance in pathogens (Hizbullah et al., 2015).

Nosocomial infections are very common and lethal in the newborns, adults and children. This greater susceptibility to infections can be explained by the immune- suppressed state of this period of life (Shitaye, 2008). Almost 7 to 73% of neonatal mortality is associated with hospital infections (Carr, 2000; Campbell, 2000).

In all over the World, Methicilin Resistant Staphylococcus aureus (MRSA) is the major source of nosocomial infections. Community and hospital-associated infections are caused by S. aureus. Methicillin-resistant strains of S. aureus cause more than 50% infections. Permanent and transitory types of S. aureus are present in nosocomial environment (Al Tawfiq, 2006; Yao et al., 2010; Sayyad et al., 2016).

P. aeruginosa infection is a highly destructive gram-negative neonatal infection. The second highly isolated glucose non-fermenter Bacillus is P. aeruginosa (Elgderi et al., 2006; Giamarellou and Kanellakopoulou, 2008). In many countries, S. typhi, gram- negative facultative intracellular anaerobes belonging to Salmonella spp., have emerged as an important cause of nosocomial infection. Over the last decades, it has been reported that amoxycillin-cephalosporin resistant Salmonellae caused nosocomial outbreak (Mache, 2002). E. coli and Salmonella are the species of family Enterobacteriaeceae which inhabit the exoskeleton of cockroaches. E. coli can reside in food and water for a long time. It can grow best at 37ºC which is the body temperature of a healthy human (Masood et al., 2014).

Shigella species inhabit gut which is a suitable environment for their survival and dispersion because in gut of cockroaches, species of Shigella may survive for a long time. As a host, cockroaches discharge S. flexneri with their feces for long time and are found as a considerable threat for consumers and community (Solomon et al., 2016).

Proteus spp. is a gram-negative facultative anaerobe and proteolytic rod which are human opportunistic pathogens and found in urine, wound and other clinical sources. Recent investigations reported that the genus Proteus comprises of P. vulgaris, P. penneri, P. hauseri and P. mirabilis. Cockroaches and flies are considered as common vector of different microorganisms including P. vulgaris strains and P. mirabilis 7

because they carry them on their bodies and disperse them as spoiling food or contamination and also include infections to humans (Drzewiecka, 2016; Wannigama et al., 2014).

In houses, cockroaches may bite at the lips and the sharp sides of the mouth of the sleeping children or bedridden people, especially the children who regurgitate milk, causing lesions called Herpes blattae. The growth and development of cockroach populations in food preparation areas and multi-family houses are mainly due to domestic contamination and poor sanitation. In houses, cockroaches are considered as the positive health risk for the human beings (Mpuchane et al. 2006). It has been found in United States that 23% to 60% of urban residents are sensitive to cockroach allergens like asthma and children as young as 10 months are sensitive (Litonjua et al., 2001).

In immune-compromised patients, especially those who stayed in hospitals for a long time nosocomial infections are caused by nosocomial bacterial and fungal agents. Outbreaks of nosocomial mycosis, was caused by exposure to medically-important fungi within the hospital environment (Bouza and Munoz, 2004). Patients that are admitted in intensive care unit (ICUs) may suffer serious health care associated infections mostly due to the fungal Aspergillus spp. and Candida spp. infections (Bouza and Munoz, 2008).

Medically important fungi, such as Rhizopus, Alterneria, Candida, Mucor and Aspergillus spp. were found and isolated from cockroaches in hospital wards and residential areas (Fotedar and Banerjee, 1992). Nosocomial fungal infections are caused by 2 yeast and 9 filamentous species of fungi that were isolated from the cockroaches in various studies (Motevali Haghi et al., 2014).

In patients of lungs disorders and bone marrow transplant recipients, Aspergillosis is very common. Aspergillus spp. have been isolated from 36% patients and caused high death rate. Presence of Aspergillus niger (A. niger) on some fruits and vegetables caused Black mold disease. It has been found evenly in soil and caused otomycosis among Nigerians (Issac et al., 2014). Mucor is a microbial genus of approximately 3000 species of mold that are common in soil, plants surfaces, digestive system and rotten vegetables matter. Human zygomycosis is caused by Rhizopus species which is present on organic substrates (De Hoog et al., 2000). In hospitalized patients, Candida 8

spp. were considered as the fourth most common organisms that were recovered from culturing of their blood (Nabili et al., 2013).

In neonatal units, fungal infections are more prevalent as compared to isolates of gram-negative bacteria. The main factors of infections in hospitals are prolonged exposure to antibiotics, tracheal intubation, and intravenous infusion of lipids and parental hyper ingestion of antibiotics during pregnancy. Different Candida strains like albicalis, tropicalis and parapsilosis were named so for most hospital infections it causes. Therefore, the most common fungal infection in human is fungemia. Fungi can cause endophtalmitis, spleen and kidney abscess, meningitis and osteomyelitis while, 25 to 50% death rates are also associated with fungi in developing countries (Davies and Gibbs, 2001).

Not only bacterial and fungal contaminants are carried by cockroaches but also protozoan parasitic, eggs or helminthes worms are also carried by cockroaches in human dwellings. Medically important parasites, such as protozoan parasites and gastro-intestinal helminthes present on the external parts of cockroaches were isolated (Alzain, 2013). By feeding on triatomine vectors of Chagas disease, cockroaches can contribute towards the transmission of that disease (Al-Mayali and Al-Yaqoobi, 2010).

Female cockroaches are more vectorial than the male. This attribute can be explained as they move more than the male in search of food and sites to lay their eggs. As they roam, they come in contact with contaminated material, making them more harmful when contaminated with pathogens (Hamu et al., 2014; Ojianwuna, 2014).

Predominant helminthes carried by cockroaches are Ascaris lumbricoides ova (A. lumbricoides), Trichuris trichiura (T. trichiura), Strongyloides stercoralis (S. stercoralis), rhabditiform larva and Taenia spp. egg. The protozoans include Enterobius vermicularis (E. vermicularis) eggs, Schistosoma mansoni (S. mansoni) ova, Schistosoma haematobium (S. haematobium) ova, Balantidium coli (B. coli) trophozoites, Hookworms, Entameba coli (E. coli) cysts, Entameba histolytica (E. histolytica) cyst, Endolimax nana (E. nana) cysts, Blastocystis hominis (B. hominis), coccidian parasites: Isospora belli (I. belli) oocysts, Cyclospora cayetanensis (C. cayetanensis) oocysts, Cryptosporidium parvum (C. parvum) oocysts, Chilomastix mesnilli (C. mansilli) cysts, and Iodamoeba butschlii (I. butschlii ) cyst, Ancylostoma 9

duodenale (A. duodenale) larvae, Moniliformis moniliformis (M. moniliformis), Nyctotherus ovalis (N. ovalis) (Ghosh and Gayen, 2006; Salehzadeh et al., 2007; Ajero et al. 2011; Al-Bayati et al., 2011; Chamavit et al., 2011; Pennapa et al., 2011; Bala and Sule, 2012; Nedelchev et al., 2013; Iboh et al., 2014; Isaac et al., 2014; Yaro et al., 2015; Sia Su et al., 2016).

On external body of cockroaches the most common protozoan E. histolytica is present that has resistant cyst wall and causes amoebiasis. E. histolytica continued its life in outer environment and afterwards was transmitted to humans by cockroaches (Bala and Sule, 2012). On the external body surface of cockroaches B. coli trophozoite can reside on cattle’s during foraging of food and occasionally human faeces. In this way, opportunity for them to spread pathogenic agents to human is avoidable (Chamavit et al., 2011; El-Sherbini and El-Sherbini, 2011).

Recovery of T. trichiura and A. lumbricoides ova from the external washing showed that cockroaches are seriously involved in the epidemiology of soil transmitted helminthes (STH). The causative agents of human helminthiasis are T. trichiura, A. lumbricoides, S. haematobium and S. mansoni (Etim et al. 2013). Isolation of E. vermicularis signifies the clear contact of cockroaches with waste dispoal and clothes of patients (Chan et al., 2004).

Amoebiasis, Ascariasis and Giardiasis are the diseases that are caused by pathogenic helminths and protozoans are usually transmitted by cockroaches. In the infected individuals these are also responsible for liver failure, stunted growth, chronic diarrhea and intestinal disturbances (Adeleke et al., 2012). Cockroaches that are present in toilet had more parasitic load because they can be easily exposed to and contaminated by faecal matter (Ojianwuna, 2014).

In the previous studies, antibiotics resistant bacteria were not only found in cockroaches collected from the houses but also from hospitals. So, at both locations the cockroaches have equal potential to cause outbreaks of infectious diseases. E. coli, S. aureus, Acinetobacter baumannii (A. baumannii), Streptococcus pneumoniae (S. pneumoniae), P. aeruginosa, Enterobacteriaceae, enterococcal isolates, Haemophilus infuenzae (H. influenza), K. pneumoniae, β-hemolytic streptococci, coagulase-negative staphyloccocci and many other isolates are resistant against commonly used antibiotics in hospitals. Various bacterial isolates from P. americana, 10

S. longipalpa and B. germanica frequently exhibit either single drug resistant or multidrug resistant commonly (Oliva et al., 2010; Akinjogunla et al., 2012; Pai, 2013; Brown and Alhassan, 2014; Vazirianzadeh et al., 2014; Wannigama et al., 2014).

Over 2500 years ago, antimicrobials were used to control infectious diseases. Therefore, excessive use of antimicrobials has resulted in multi resistant strains, a serious public health problem (Hall, 2004; Prado et al., 2006). A combination of restricted antibiotic uptake through the outer membrane and a variety of other energy dependent mechanism accomplished antibiotics resistance in bacteria. Pre-incubation with antibiotics have many effects on bacterial isolates including osmotic stress response and reduced bacterial adherence to media, induction of biofilm form of growth and changes to hydrophobicity (Jabber et al., 2015).

Microbial resistance to antibiotics, a potential health hazard is associated with the R- plasmids. In natural ecosystem, the bacteria that carrying R-plasmids have a better chance of survival and selection as compared to those strains that lack R- plasmids. The bacteria become temporarily inactive when encountered with insufficient and sublethal doses of disinfectants. However, after removing the traces of disinfectants the bacteria can recover from a drastic inactivity (Tewari et al., 2003).

Multi drug resistant (MDR) strain could arise in a bacterial cell due to the accumulation of many resistant genes or presence of such gene that express activity of multidrug efflux pump which extrude all drugs instantly after application. Total resistance for penicillin was observed for Gram-positive bacteria such as S. aureus and coagulase negative staphylococci (CNS) species but no resistance was observed for methicillin and vancomycin. Moges et al. (2016) confirmed that all isolates showed high resistance toward cotrimoxazole followed by amoxicillin, clavulanate and tetracycline; however low resistance was observed for ciprofloxacin.

Antibiotics resistance virulence factors are encoded by plasmids, bacteriophages and chromosomes. Plasmid-encoded β-lactamases like TEM-1, TEM-2, and SHV-1 expressed by E. coli. The commonest factor conferring resistance toward penicillin is TEM-1 gene. In E. coli the TEM-1 gene is responsible for upto 90% of ampicillin resistance. Hydrolysis of penicillins and early cephalosporins like cephaloridine and cephalothin was supported by activation of TEM-1 gene (Khalaji et al., 2013; Ghasemi-Dehkordi et al., 2015). 11

In ordinary population, the factors that contribute in the rate of antibiotics resistance is the host ecology and environment. As compared to E. coli, different trends for resistance are presented by Salmonella strains. Salmonella living in non-mammalian hosts in their ordinary environments encountered less with commercially used antibiotics. On the other hand, E. coli exhibit more antimicrobial resistance because they are distributed irregularly on non-mammalian and mammalian hosts and are mostly expose to commercial antibiotics (Asrat, 2008).

In houses and hospitals, various disinfectants are applied on floor and laboratory bench-tops to control nosocomial infections causing agents. Appropriate cleaning methods are used in hospitals to ensure safety of rooms and equipments for patients. The cleaning procedures vary between hospitals and even from person to person. Chlorine, phenol, iodine and alcohol are most commonly used disinfectants in hospitals (Abreu et al., 2013).

In hospitals the inanimate surfaces play an important role in transmission of nosocomial pathogens. Although in hospitals, room of previously admitted infected patients may be a risk factor for next patient to be admitted there as both Gram- positive and Gram-negative bacteria can be transmitted by surfaces (Carling, 2013).

The destruction of vegetative pathogens from an inert surface is usually carried by disinfection destruction of pathogens at living tissue is called antisepsis and the chemical is then called as antiseptic (Willey et al., 2008). The disinfectant destroys the pathogens either by coagulating the protein of bacteria or by destroying its cell membrane and removal of a sulphonhydric group from the (Omoruyi and Idemudia, 2011). Disinfection of cells sometimes results in destruction of nucleic acid, lipids or proteins also (Olowe et al., 2004; Okore et al., 2014).

Direct influence of a surface disinfectant in destruction of pathogens during outbreak at an area cannot prove because for controlling an outbreak many interventions reinforce in parallel collectively to control an outbreak. By introducing a bundle of surface disinfections, improved staff training, improved communication and patients screening a K. pneumonia an outbreak could be controlled (Cioboraro et al., 2011).

Dettol® is mostly used in homes and health care settings for disinfecting of environmental surfaces, skin, objects and equipments. By applying disinfectants the 12

microorganisms colonizing the surfaces and skin are greatly reduced. Chloroxylenol have antimicrobial properties and is the chemical constituent of Dettol® and other chlorinated phenol (Mellefont et al., 2003). Manufactures of Dettol® suggested that in skin disinfection biocides, such as the (chloroxylenol), purit (5-chlorohexidine gluconate), septol (5-chloro 2-hydroxy diphenyl methane) and parozone (Sodium hypochloride should be used. Mechanism of action of membrane active agent Chloroxylenol (Dettol®) is just like phenol. In bacterial cell the membrane active agent is absorbed and caused growth retardation. The absorbed quantity of chloroxylenol exhibits its bactericidal activity. The rapid destruction of structure and functions of cell membrane results in broad-spectrum cytoplasmic constitutent’s loss of bacterial cell. This membrane damage and metabolites loss is unable to be repaired by bacterial cell (Gupta et al., 2004).

The powerful bactericidial, mycobactericidal, sporicidal, fungicidal and viruscidal activities are displayed by Glutaraldehyde, a dialdehyde. In bacterial cell, the mechanism of action is based on its interaction with amino group or protein. A sufficient sporicidal effect is achieved by 2% solution of Glutaraldehyde. For surface disinfection Hydrogen peroxide is normally used. Hydroxyl free radical (•OH) produced by hydrogen peroxide act as an oxidant that will act on cell components like DNA, lipids and proteins. Its mechanism of action is to target the visible sulfhydryl groups and double bond. Alcohols exhibit rapid broad-spectrum antimicrobial activity against vegetative bacteria (including mycobacteria), fungi and viruses but no sporicidal activity was observed with alcohols. 60-90% range is optimum for the activity of alcohol, while activity of alcohol also reduced at concentration below 50% (McDonnell and Russell, 1999; Ganavadiya et al., 2014).

Infection rate can be decreased by improvement in surface disinfection, monitoring the disinfection process and feedback to cleaning staff instead of just reporting a level of contamination for an area (Meyer et al., 2015). The cell wall permeability to biocide exhibits the level of biocide sensitivity among bacteria. While, permeability of cell wall to biocides is based on composition of cell wall and physiological adaptations of the microorganism to outer environment (Lambert, 2002). Due to difference in membrane structure and many methodological problems and high intrinsic resistance the activity of biocides against microorganisms is not always the 13

same. Contamination of disinfectants can arise from inappropriate techniques, contaminated equipment and improper storage of disinfectants (El-Mahmood and Doughari, 2008).

Bacteriostatic activity of a given disinfectant is assessed by Minimum Inhibitory Concentration (MIC) while calculation of bactericidal activity under same condition is called Minimum Bactericidal Concentration (MBC). The activity of Dettol® is badly affected by tap water and serum contamination during dilution. The effective concentration of the antimicrobial agent available to microorganisms is reduced by serum. Presence of impurities like Mg2+, Fe2+ and Ca2+ traces in tap water reduce effective concentration of Dettol® by reacting with its chloroxylenol component results in increased MBC concentration (El-Mahmood and Doughari, 2009).

Reduction in transmission of pathogenic bacteria can be achieved by controlling the arthropod vector. In addition, a particular infection cannot be arbitrated whether it was caused by cockroach or house fly vector; however, presence of similar resistant bacteria on these insects and a patient who presents an infection in the same geographical areas can be related. The degree of virulence of bacteria and therapeutic possibilities in the case of infection is determined when bacterial resistance to antibiotics is known. Unhygienic and filthy habitats of cockroaches are an important factors that contributes to the diversity of cockroaches. Dispersion of cockroaches in our urban environment has a great impact in transmission of nosocomial microbes. In Pakistan awareness to this problem has been minimal or none as no previous research has been conducted on the carriage of antibiotic resistant pathogenic bacteria by cockroaches in hospitals and houses of Lahore, Pakistan. Although an increasing frequency of antibiotic resistant microbial strains has been reported from all over the world including Lahore yet role of cockroaches in dispersal of these resistant strains is not evaluated in Lahore. Hence an investigation was conducted to determine frequency of resistant and susceptible bacteria from cockroaches and elucidate their role in spreading health-associated problems in human populations.

AIMS AND OBJECTIVES

This study was conducted at Entomology Research Laboratory in the Department of Zoology, Lahore College for Women University Lahore, Pakistan. Microbial screening was done for domestic cockroaches collected from hospitals and houses of urban area of Lahore. Resistance against antimicrobials and commonly used disinfectants was determined for bacterial isolates that illustrate the role of cockroaches as mechanical vector of resistant pathogens causing nosocomial infections and food borne illness, unhygienic conditions of collection sites. The results of this study will hopefully generate awareness in people about role of cockroachis in spreading various diseases.

Objectives of this research work were:

• Taxonomical identification, diversity and population range of cockroaches collected from hospitals and houses of Lahore in four different seasons.

• Isolation and identification of parasites, fungi and bacteria from external body surface and gut tract of cockroaches to assess the presence of medically important pathogenic microbial fauna. • Investigate the efficacy of commonly used disinfectants on bacterial isolates and their resistance.

• Determine the susceptibility of most resistant bacterial isolates to 5 commonly practiced antibiotics.

• Band pattern analysis of whole cell protein fractions of most resistant bacterial isolates.

LITERATURE REVIEW

Cockroach is a synanthropic nuisance pest in human dwellings and may serve as a mechanical vector for pathogenic microbes. Cockroach feces and external body parts have been implicated in transmission of various pathogens and allergens to humans. Moreover, pathogenic bacteria isolated from cockroaches have acquired resistance to commonly used antibiotics and disinfectants in hospitals and houses. These resistant isolates cause outbreaks of nosocomial infections in hospitals and food-borne illnesses in houses. This study explored the role of cockroaches in biological and mechanical transmission of pathogenic multidrug resistant bacteria, fungal spores and parasitic contaminants in hospitals and houses of Lahore, Pakistan

Cockroaches are abundant almost throughout the year, but are fewer in February and March than at other times of the year with their numbers increasing fairly constantly from April to September. Eads et al. (1954) investigated role of cockroaches in the spread of gastro-enteric diseases in manhole closed sewerage system of Texas. They found cockroach species of P. americana, P. fuliginosa (Serv.), B. orientalis L. and P. pensylvanica (Deg.) and studied them for bacterial contaminants. They isolated 9 species of Salmonella from P. americana but no Salmonella was recovered from B. orientalis and B. germanica (L.) medium to heavy infestations of P. americana were found in more than half of the manholes inspected by Eads et al. (1954).

Rueger and Olson (1969) surveyed 19 cities throughout the United States for cockroaches and isolated 2 species of Salmonella; S. anatum and S. oranienburg from P. americana L. They isolated an enterotoxigenic strain of S. aureus from fecal pellets of cockroaches. In an experimental study, S. aureus was recovered from fecal pellets of P. americana 3 days after the infective feeding; however, S. typhosa occurred 5 to 6 days and S. flexner from 2 to as long as 11 to 13 days. The survival duration of the microorganisms studied by spreading roach pellets on different foods and glass were observed and lasted as long as 3 years 3 months on corn flakes, more than 4 years 3 months on crackers and more than 3 years 8 months on glass slides.

Wishart and Riley (1976) compared efficacy of the disinfectants purit, septol and z- germicide containing chlorohexidene gluconate as the main constituent and reported

that purit was the most contaminated, with 30% of the samples yielding bacterial growth. Since their preparation of the dilutions of the disinfectants used was made in a similar way, it could be assumed that the other two disinfectants, especially septol with only 10% and z-germicide with 15% would also be contaminated at the same level. The observed differences in MIC may be due to the low concentration of chlorohexidine in the formulation of purit. Previously, outbreaks of Pseudomonas maltophilia (P. maltophilia) infections associated with contaminated solutions of chlorohexidine gluconate and cetrimonium bromides have reported from some Australian hospitals.

Cockroaches are not only a nuisance but are also a serious health hazard. In 1976 at Farmingdale in New York State, Frishman and Alcamo (1977) carried out an investigation on domestic cockroaches [B. germanica (L.), P. americana (L.) and B. orientalis L.] as vectors of bacteria capable of causing food poisoning and other diseases in man. Nymphs and adults of all 3 species were proven to carry at least 1 type of bacterium capable of causing food poisoning. All species carried Staphylococcus in medium to high concentrations. High concentrations of E. coli were discovered only from Blattella whereas Salmonella was lowest on or in Blatta. Streptococcus was obtained only from 2 hospitals sites.

Cruden, and Markovetz (1987) investigated P. americana for bacterial population and isolated over 100 species of bacteria from P. americana. The higher microbial isolation from alimentary tract proved that it a potential environment for the development of the microbial communities. Allen (1987) repeatedly isolated Mycobacterium tuberculosis (M. tuberculosis) from homogenized fecal pellets of cockroaches. Faeces remained positive both microscopically and on culture even after storing for 8 weeks at room temperature. These nocturnal omnivorous insects which frequently infest hospitals and laboratories may interact with patient faeces or clinical samples and carry this bacterium along with them.

Le Guyadera et al. (1989) conducted a study in a main hospital in Rennes (France) on the distribution and associated bacterial flora of the cockroach (S. longipalpa). Wild cockroaches carried contaminant flora acquired from particular environments. The

diversity of carried bacterial species revealed a proximity factor between continguous floors of the building and cockroaches that forage from one floor to the other.

Fotedar et al. (1991) investigated hospital inhabiting cockroaches as vectors of drug- resistant Klebsiella spp. at the All India Institute of Medical Sciences (AIIMS) hospital during Nov 1985 to April 1989. Klebsiella spp. Mostly K. pneumoniae was isolated from 28.3% of hospital cockroaches and 28.1% of infected wounds of patients. Most of Klebsiella isolates from patients (96.3%), and hospital cockroaches (85.9%) showed multiple drug resistance to four or more antimicrobials. Whereas, all strains of Klebsiella spp. isolated from the control cockroaches were sensitive. This difference was statistically significant. Some strains of Pseudomonas spp. were often isolated from insects caught in different environments, including hospital areas.

Emori et al. (1991) described the prevalence of MRSA in hospitals. Prevalence of MRSA accounts for <10% of all S. aureus isolates, whereas in other facilities they account for upto 65%. Prevalence of MRSA varies considerably from one region to another and among hospitals in the same city. National nosocomial infection surveillance system (NNIS) reveals methicillin-resistant Staphylococcus aureus (MRSA) accounts for upto 40% of nosocomial S. aureus infections in large hospitals and 25-30% in smaller hospitals. Richet et al. (1996) reported 1.25% prevalence of MRSA in 27 hospitals of France, whereas Beardi-Grassias et al. (1996) reported 31.8% in another hospital in the same area.

Lelievre et al. (1999) investigated incidence of isolation of Gentamicin sensitive MRSA (GS-MRSA) and found a steady increase over a 7 year period to represent in 1998, 46.8% to 94.4% of all strains. Most MRSA strains are multi-drug resistant. Resistance to erythromycin and clindamycin was very common and many strains were resistant to gentamicin, tobramycin and ciprofloxacin as well. Previous studies had recovered some strains resistant to methicillin but susceptible to gentamicin.

Cotton et al. (2000) described Infections in basically two groups; the infections that arise up to 48 hours of life are deliberated hospital infections while hospital infections originated by the environment are those that occur after 48 hours of life. The US Center for Disease Control and Prevention (CDC) reported in a study that hospital

associated infections (HAIs) appeared up to 48 hours after hospital discharge or during hospitalization. Some trans-placental infections (toxoplasmosis, HIV infection, rubella, hepatitis B, cytomegalovirus infection and herpes simplex) are exceptional.

Cotton et al. (2000) conducted a study in a neonatal unit infested with cockroaches. Extended spectrum beta-lactamase-producing K. pneumoniae isolated from cockroaches were not different from those bacteria colonizing infants or causing clinical disease. It showed that cockroaches are possible vectors of bacterial pathogens in the hospital environment.

Chang et al. (2000) conducted a study in medical centers of Taiwan and observed that more than 30% of S. aureus, S. pneumonia, Enterobacteriaceae, P. aeruginosa, Acinetobacter baumannii (A. baumannii), H. infuenzae, CNS, β-hemolytic streptococci, viridans Streptococci and Enterococcal isolates were found to be resistant to commonly used antibiotics.

Mayfield et al. (2000) described that after ceasing surface disinfection while maintaining all other measures, the MRSA rate rose again to its previous value. By introducing sporicidal instead of just bactericidal surface disinfection, the rate of C. difficile-associated diarrhea was significantly reduced. Returning to bactericidal surface disinfection, the rate rose to its former value.

Existence of cockroaches in hospitals with highly infected pathogens, such as bacteria like Klebsiella and E. coli can cause bacterial epidemics to all wards from the infection ward. Some people are allergic to excrement of cockroaches due to the presence of proteins in their feces (Adibfar, 2000). The number of areas containing waste refuse and human excreta provide best environment for population explosion of synanthropic insects like house flies and cockroaches (Fotedar, 2001). Vatani (2001) isolated bacteria; E. coli, Klebsiella, Staphylococcus, Pseudomonas, Shigella and Salmonella from P. americana in Tehran hospital, Iran.

Wallace (2001) tested the antimicrobial efficiency of PHMB against gram-positive S. aureus and gram-negative K. pneumoniae. PHMB was applied to cotton fabric, and the treated fabric was subjected to 1, 5, 10, and 25 laundering cycles using Tide detergent before antimicrobial tests. The results showed that PHMB reduced S.

aureus by 98 % after more than 10 laundering cycles and caused >99 % reduction of K. pneumonia after 5 laundering cycles and more.

Czajka et al. (2003) identified bacterial flora from external parts of German cockroaches trapped from hospitals. They isolated 80 strains of bacteria, among which 34 were Gram-positive cocci and 31 were Gram-negative rods. One isolated strain of Citrobacter freundii (C. freundii) and two strains of Serratia liquefaciens (S. liquefaciens) showed ESBL mechanism of resistance and extended level of AmpC- type beta-lactamases. Two Staphylococcus strains; Staphylococcus epidermidis (S. epidermidis) and Staphylococcus equorum (S. equorum) were resistant to erythromycin and clindamycin; isolated strains of P. aeruginosa and P. putida from the hospital cockroaches were resistant to penicillins, cotrimoxazole and trimethoprim.

Gliniewicz et al. (2003a) carried out a study on cockroach in a neonatal intensive care unit of a hospital and isolated Enterobacter spp., a common cause of neonatal sepsis. Most common Enterobacter species carried by the cockroaches was Enterobacter cloacae (E. cloacae) carried by cockroahes. Hervas et al. (2001) conducted a study on outbreaks of E. cloacae sepsis in neonatal ICUs and isolated high loads of E. cloacae from cockroaches. Isolation of E. cloacae from external surface of cockroaches collected from neonatal ICU indicated cockroaches as the possible source of outbreak of the infection. Roy et al. (2002) isolated multi-drug resistant Enterobacter spp. from cockroaches. Isolation of high sensitivity of Enterobacter spp. to highly potent floroquinolones (Ciprofloxacin and Norfloxacin) revealed multi-drug resistance of the cockroaches to the bacteria.

Gliniewicz et al. (2003b) inspected drug resistance of microbial contaminants of German cockroaches caught from hospitals in Poland. Nosocomial infections causing bacteria were isolated from the external surfaces of these cockroaches; several bacterial isolates showed resistance to antibacterial drugs and were insensitive to chemical disinfectants used for surface disinfections.

Tewari et al. (2003) studied drug resistant Enteropathogenic E. coli (EPEC 086 serotype) isolated from contaminated piped drinking water supply (Fecal coliform

160/100ml) for effects of disinfectants (chlorine and Ultraviolet B) on stability and transmissibility of R-plasmid of 3.7 kb size. UVB did not affect the pattern of transmissibility of R-plasmid but enhanced frequency of transfer. UVB irradiation induced breakage of large plasmid DNA molecule into smaller increasing its mobility and enhancing rate of transfer. Sodium hypochlorite (chlorine) improves the permeation of the bacterial membrane without causing lysis. After exposure to sublethal doses of disinfectants, complete elimination of resistance to streptomycin and Cr was observed. Partial loss of resistance to Hg due to chlorine was detected.

Cockroaches can play a role in bacterial transmission through their cuticles. Chaichanawongsaroj et al. (2004) surveyed three different areas such as hospital, food-handling establishments and human dwellings for pathogenic gram-negative bacteria carried on the cuticles of cockroaches. The most frequent potentially pathogenic bacteria found were E. coli, K. pneumoniae, C. freundii and E. cloacae comprising 58% of all bacteria identified by the authors. The number of pathogenic and potentially pathogenic bacteria was similar in hospital areas and food-handling establishments, while human dwellings supported a poorer bacterial flora; E. coli, K. pneumoniae and E. cloacae were dominant species in hospital areas, while in food- handling establishments and human dwellings E. coli, K. pneumoniae and C. freundii predominated.

In a study Pai et al. (2004) isolated 23 bacteria and 12 fungi from B. germanica. They reported multi-drug resistance with resistance to Ampicillin (13.7%-100%), Chloramphenicol (14.3%-71.4%), Tetracycline (14.3%-73.3%), and Trimethoprim- Sulfamethoxazole (14.3%-57.1%) in 2 gram-positive and 5 gram-negative bacteria.

Pancer et al. (2004) studied the susceptibility (MIC) of 21 Gram-negative bacilli isolated from cockroaches in hospitals to disinfectants (glucoprotamine, sodium dichloroisocyanurate, potassium persulfate). Effectiveness of these disinfectants against selected bacteria revealed that glucoprotamine showed the highest activity against Gram-negative bacteria. MIC values for glucoprotamine were 16-64 times lower than MICs for sodium dichloroisocyanurate and 4-32 times lower than MICs for potassium persulfate. The effectiveness of disinfectants containing potassium persulfate or sodium dichloroisocyanurate was 100% tested by carrier method.

Glucoprotamine was ineffective against 2 out of 9 strains (18%): E. cloacae and S. marcescens. It was found that disinfectants were more effective against Gram- negative bacteria in carrier methods than for biofilm forming bacteria; 86% of bacteria growing 5 days on a catheter were resistant to glucoprotamine (5200 mg/L) or potassium persulfate (4300 mg/L) while, 66.6% of tested bacteria were resistant to sodium dichloroisocyanurate (1795.2 mg/L). The effectiveness of disinfectants to biofilm forming bacteria showed the highest effectiveness with sodium dichloroisocyanurate and lowest with glucoprotamine.

In Pakistan little research has been carried out on microbial screening of cockroaches. Mlso et al. (2005) investigated species dynamics of cockroaches as well as prevalence of various bacteria in a tertiary care hospital of Rawalpindi. American cockroach was the highest (73%) in numbers at all the locations, followed by the oriental cockroaches (18%) and then the German cockroaches (9%). Many bacterial species were isolated including: Proteus spp. 11.5%, Enterococcus spp. 13.4%, Citrobacter spp. 11.3%, E. coli 9.7%, K. pneumoniae 12.8%, Bacillus spp. 6.9%, Enterobacter spp. 8.0%, Pseudomonas spp. 8.0%, P. aeruginosa 5.7%, Klebsiella oxytoca (K. oxytoca)1.8% Providencia spp. 3.4%, S. marcescens 4.7% and Staphylococcus spp. 2.3%. Enterococcus spp. was the most abundant bacterial isolate of P. americana in the hospitals. Cockroaches collected from Medical ward-2, Medical ward-10 and Medical ward-16 were 16%, 13% and 22% respectively. Highest population of P. americana (95.4%) were found in ward-16 while Medical ward-2 had 93.7%. Both wards were close to each other and lacked proper sanitary conditions. However, mess of nearby food materials (particularly in pantry) provided excellent feeding and breeding site for cockroaches.

Pai et al. (2005) collected P. americana and B. germanica from 40 households in Kaohsiung City and Kaohsiung County, Taiwan, and investigated for microbial contamination. Cockroach infestation was found in 50% of the studied households and 226 cockroaches (123 P. americana and 103 B. germanica) were trapped. P. americana occurred more often in the kitchen (70.7%), whereas B. germanica in the storage room (51.5%) and kitchen (36.9%). There was no significant difference between the percentages of P. americana (99.9%) and B. germanica (98.0%) carrying

bacteria. A total of 25 species of bacteria was isolated from P. americana and only 21 from B. germanica. Resistance to antibiotics was found in S. aureus, Enterococcus spp., P. aeruginosa, K. pneumoniae, E. coli, S. marcescens, and Proteus spp. isolated from the cockroaches.

Saira (2005) collected cockroaches from different areas of Lahore for microbial screening. Cockroaches were fed with simple boiled eggs and infected eggs. Ten genera and 9 species of microbes were isolated including: Acinetobacter anitratus (A. anitratus), Aerococcus viridans (A. viridans), E. coli, Flavobacterium spp., Micrococcus varians (M. varians), Neisseria gonorrhoeae (N. gonorrhoeae), Salmonella typhimurium (S. typhimurium), S. aureus, Streptococcus faecalis (S. faecalis) and P. aeruginosa.

Tatfeng et al. (2005) investigated involvement of cockroaches in the transmission of tropical diseases in houses with pit latrines and water system in Ekpoma, Africa, between January and June 2005. The bacterial, fungal and parasitic isolates were identical irrespective of the site and included: E. coli, K. pneumoniae, P. vulgaris, P. mirabilis, C. freundii, E. cloacae, Salmonella spp., P. aeruginosa, S. marcescens, S. aureus, S. faecalis, S. epidermidis, Aeromonas spp., Candida spp., Rhizopus spp., Aspergillus spp., Mucor spp., cysts of E. hystolitica, oocysts of C. parvum, C. cayetenensis and Isospora belli, cysts of Balantidium coli, ova of A. lumbricoides, A. duodenalae, E. vermicularis, ova T. trichura, larva of S. stercoralis. Cockroaches trapped from houses with pit latrines had mean bacterial and parasites count of 12.3 × 1010 org/ml and 98 parasites/ ml respectively, while 89.5 × 107 org/ml and 31 parasites/ml respectively, for houses with water system. A bacterial count of 78.9 × 107 org/ml was recorded from cockroaches trapped from the kitchens of houses with pit latrines while 23.7 × 106 org/ml for kitchens of houses with water system.

Elgderi et al. (2006) conducted study in the Neonatal Intensive Care Unit (NICU) in Tikur Anbessa University Hospital, Addis Ababa, Ethiopia. Pseudomonas spp. was one of the common blood culture isolate from sick newborns. There was a multi-drug resistance pattern among all the isolates of Pseudomonas spp. Sensitivity for Floroquinolones (Ciprofloxacin and Norfloxacin) was found to be 95%, whereas sensitivity to penicillin and gentamicin was 0% and 21% respectively.

Lemos et al. (2006) isolated Candida spp. and Rhodotorula yeast from both B. germanica and P. americana. Among Candida species, Candida albicans (C. albicans) was the most commonly isolated and was responsible for the majority of nosocomial infections. However, many non-albicans species, such as Candida glabrata (C. glabrata), Candida parapsilosis (C. parapsilosis), Candida tropicalis (C. tropicalis) and Candida guilliermondii (C. guilliermondii) had emerged as important pathogens in suitably debilitated individuals. C. glabrata was frequently isolated from older patients, patients with cancer and patients treated with vancomycin. C. parapsilosis was an important cause of candidemia in the neonatal population and transplant recipients. C. parapsilosis was the most common Candida spp. isolated from the hands of health care workers. Candida spp. resistant to fluconazole, such as C. guilliermondii had been associated with nosocomial outbreaks. Rhodotorula spp. caused nosocomial endophthalmitis and meningitis in human immunodeficiency virus (HIV) infected persons. However, various mold species were isolated from external surface of cockroaches, such as Fusarium spp., Penicillium spp., Geotrichum spp., Alternaria spp., Cladosporium spp., Trichoderma spp. and Mucor spp.

Bacteriological investigations were carried out by Mpuchane et al. (2006) on cockroaches trapped from the kitchens and toilets of 3 localities in Gaborone, Botswana. The cockroaches trapped from the toilets had higher counts than kitchens. Low number of spore formers was observed in most samples. However, Bacillus cereus (B. cereus) was found in some of the cockroaches at much lower numbers. 70–98.3% of the cockroaches had coliforms but E. coli was only found in 5–6.5% of the cockroaches. Collectively, 70 species of bacteria were isolated from the surface and fecal pellets. Majority of the bacteria isolated from the surfaces were gram-negative while mostly gram-positive were isolated from fecal pellets. Most common isolates were Pseudomonas and Serratia followed by Enterobacteriaceae family. Bacillus spp. was dominant in fecal pellets followed by some members of Enterobacteriaceae. Pathogenic isolates including Salmonella, Shigella and B. cereus, opportunistic pathogens like species of Pseudomonas, Klebsiella and Vibrio and food spoilage bacteria such as species of Enterobacter, Citrobacter, Escherichia, and Erwinia were also found.

Previous studies described many bacterial strains isolated from arthropods caught in the hospitals or nearby areas were resistant to antibiotics and chemotherapeutics. They may had different mechanisms of resistance including: ESBL (extended spectrum of beta lactamases) and AmpC (AmpC beta lactamases) (Gram – negative rods), methicillin resistance (MRSA, methicillin resistant S. epidermidis), MRCNS and MLSb (resistance to macrolides, lincosamides, streptogramin B).

Pancer et al. (2006) isolated E. cloacae strains from the external body surface of the German cockroach B. germanica L. that showed phenotypically ESBL activity besides AmpC beta-lactamases. Presence of plasmids with ESBL enzymes in the bacteria meant resistance to penicillins, cephalosporins and monobactams. On the same plasmid genes can be present, which determine resistance to other groups of antibiotics, for example amino - glycosides or sulfonamides. ESBL enzymes, which were currently detected in E. cloacae, S. marcescens, K. oxytoca, were typical for the strains of K. pneumoniae and E. coli only.

Prado et al. (2006) conducted research in health care institution in Brazil. They analyzed 103 cockroaches for microbial screening and drug resistance, and found 97% fungi, 74.6% enterobacteria and 25.40% CNS. Among the enterobacteria, 96% were resistant to gentamicin, 84% to ampicillin, 75.3% to caphalothin, 66.7% to ampicillin-sulbactam, 50% to aztreonam and 30% to chloramphenicol whereas CNS exhibited 61% resistance to oxacillin.

Stypułkowska-Misiurewicz et al. (2006) determined the vulnerability of cockroaches for patients in hospital environment. The microbiological hazard of cockroaches in hospital environment had been proven by isolation of bacterial strains well-known for hospital infections carried on the body of cockroaches. Isolated strains were resistant to several antibiotics used in hospitals. Correlation was found between resistance of cockroaches for biocides and higher infestation in the hospital environment.

Tachbele et al. (2006) studied the microbiology of cockroaches from hospitals and restaurants in Ethiopia Addis Ababa. Four sero-groups of Salmonella, Shigella, E. coli, S. aureus and B. cereus were isolated. Most of the isolates were recovered from both gut and external surfaces of cockroaches. Higher drug resistance incidence was observed in hospitals than in restaurants. No statistically significant differences were

found in the distribution of potential pathogens between source, location and body parts. Cockroaches shed the pathogenic Salmonella for 35 days after feeding and all cockroaches were dead at the end.

Salehzadeh et al. (2007) conducted research in different wards of hospitals of Hamadan Iran and discovered that B. germanica was contaminated with high bacterial load (more than 1 × 103), whereas only 2 out of 45 (4.45%) cockroaches of the control group were carrying medically important bacteria with comparable high bacterial load. Bacteriological examinations revealed that almost all test cockroaches had at least one of the following microorganisms either in their body surface or digestive tract: Enterobacter (22.6%), Klebsiella (21%), Enterococcus (17.3%), Staphylococcus (16.5%), E. coli and Streptococcus (8.3%), Pseudomonas (3%) while Shigella and Haemophilus were less than 1%. In addition their results showed that 74.4 % of the total test cockroaches harboured fungi, such as Candida (48.9%), Mucor (10.5%), A. niger (7.5%), Rhizopus (4.5) and also Penicillium (1.5%) and Aspergillus fumigatus (A. fumigatus) (1.5%). Some parasitic worms of medical importance were also isolated from the test cockroaches, but carriage rates were low.

Vahabi et al. (2007) observed microbiota present on cockroaches in two hospitals affiliated to the Kordestan University of Medical Sciences in 2003 and found that 89.8% of the total collected cockroaches were positive for bacteria.sixty seven percent of the bacteria were rcovered mainly from external body parts and the remaining from the alimentary tract. Bacterial infection distribution between two hospitals showed that the most infected cases with 97.5% belonged to Tohid Hospital with 63.3% external and 34.2% diagnosed from digestive tract of the collected cockroaches. In comparison, from Beasat hospital 47.7% of cockroaches were infected externally and just 10.5% were infected internally. The most common bacterium isolated externally from cockroaches was E. coli (76.3%) while no internal contamination was reported with this bacterium. The second most common bacterium found was Proteus spp. with 50.8% external and 32.2% internal infection.

Al-Marjani et al. (2008) isolated 30 Enterobacteria from cockroaches along with K. pneumonia, P. aeruginosa, E. coli and P. mirabilis, S. marcescens and E. aerogenes. All the isolates were resistant to erythromycin, cefazolin and amoxycillin showed

multiple resistance to antibiotics. Whereas all isolates were susceptible to aztreoname and ofloxacin and the majority of isolates were susceptible to cefoperazone (70 %) and trimethoprim (75 %).

Snoddy and Appel (2008) conducted a survey in Southern Alabama and Georgia to determine the extent of B. asahinai has expansion northwards from Florida. They concluded that visual and bucket sample population began increasing in late May and reached their zenith in late August or early September.

Kinfu and Erko (2008) assessed the role of cockroaches as potential carriers of human intestinal parasites in Addis Ababa and Ziway, Ethiopia. Microscopic examination of the external body washes of pooled cockroaches and individual gut contents revealed that the examined cockroaches were carriers of E. coli and E. histolytica/dispar cysts as well as E. vermicularis, T. trichiura, Taenia spp. and A. lumbricoides ova.

El-Mahmood and Doughari (2008) reported dettol® as a lethal agent against nosocomial infection causing microorganisms. There was no decrease in killing rate over the period of exponential death. All the isolates showed a similar response to dettol®. This factor may imply on subpopulation of cells in the cultured tests which are resistant to use dilution of dettols®. A shoulder followed by exponential death was exhibited by all survivor curves of isolates.

Oule et al. (2008) determined Phenol Coefficient (PC) values of Polyhexamethylene biguanidine for S. aureus, S. choleraesuis and P. aeruginosa and reported 7.5, 6.1 and 5 values, respectively whereas the MIC value for MRSA and E. coli was 0.04% and 0.005% (w/v), respectively, in 1.5 min.

Wishart and Riley (1976) investigated bacterial colony counts for disinfectants and found higher bacterial colony counts in tested isolates because there was a likelihood of attaining an infective dose at the site of application of an antiseptic. The total bacterial count (TBC) ranged from 7.5 x 105 cfu/ml to 6.3 x 108 cfu/ml while 102- 108 cfu/ml was reported by Oie and Samiya (1996) and Zembrzuska-Sadkowska (1995). Resistance towards disinfectants would be more evident when the disinfectants are used in the wards where compromised patients live and the resulting

nosocomial infections would be more severe. Hand washing with a contaminated skin disinfectant was proved to be dangerous for the health care staff.

El-Mahmood and Doughari (2009) reported that antimicrobial agents showed both inhibitory and lethal effects towards microbial isolates. The MIC and MBC values of dettol, purit, parazone z-germicide and septol revealed that concentration of the active ingredients in the recommended dilutions of the disinfectants was lethal to the organisms. The MIC and MBC values were higher than manufacture’s recommended values. The MIC of the disinfectants against the organisms was lesser as compared with the MBC values and increased with increase in the concentration of the agent used. The observed MIC of dettol against E. coli was (0.10 ml) and MBC (0.14 ml), the MIC of parazone against P. aeruginosa was (0.08 ml) and MBC (0.12 ml) while the MIC and MBC of purit against S. aureus was 0.08 ml and 0.14 ml, z-germicide was 0.12 ml and 0.14 ml and for the septol 0.10 ml and 0.14 ml, respectively.

El-Mahmood and Doughari (2009) inspected susceptibility of bacterial isolates to antibiotics and found E. coli was susceptible to 4 (chloramphenicol, gentamycin, streptomycin and tetracycline) out of 11 antibiotics and resistant to 7 antibiotics. S. aureus was susceptible to 5 (nitrofurantoin, tetracycline, gentamycin, ciprofloxacin, and streptomycin) antibiotics and resistant to 6. P. aeruginosa was susceptible to 6 (gentamycin, tetracycline, erythromycin, ampicilin, cotrimoxazole, chloramphenicol) antibiotics and resistant to 5 antibiotics. Tetracycline inhibited the growth of all the bacterial isolates but colistin had no inhibitory effects on any of the bacterial isolates.

Dancer et al. (2009) reported that the influence of surface cleaning on MRSA infection rate suggested that employing only one additional person dealing with surface cleaning per ward resulted in a significant reduction of microbial contamination of surfaces and the MRSA infection rate.

Xue et al. (2009) examined B. germanica in 5 habitats (a hospital, a restaurant, an office, a home, and a market) in Beijing. Cockroach densities significantly differed among the studied habitats in that order: market >Home >office >Restaurant >hospital but no significant differences were found in bacterial abundance among habitats. The bacterial abundance in the gut was significantly higher than that on the surface. There

were no significant differences in bacterial species richness observed among habitats, sex, carrying position or their interaction. Cockroach densities and bacterial abundance found in the market differed significantly from the other four habitats. The bacterial diversity was not significantly reduced in sensitive facilities, such as hospital and restaurant even though pesticide and bactericide were more frequently applied there.

Al-Mayali and Al-Yaqoobi (2010) investigated parasitic contaminants of cockroaches from different parts of Al-Diwaniya province, Iraq and isolated 7 parasitic species from the external surface and intestinal tracts representing 2 Protozoa and 5 Nematodes. The parasitic isolates included: Entamoeba blatti (E. blatti) (33%), N. ovalis (65.3 %), Hammersmiditiella diesingi (H. diesingi) (33.3 %), Thelastoma bulhoe (T. bulhoe) (51.4 %), Gordius robustus (G. robustus) (Horsehair worm) (1.3%), E. vermicularis eggs (2%) and A. lumbricoids eggs (1.3%) however no cestodes were observed. Prevalence of intestinal worms was significantly higher statistically than the Protozoa. The number of worms per cockroache ranged from 1-3 while Protozoa were 42 parasites/ml. Endoparasites were higher in number than the ectoparasites and intestinal worms were significantly higher statistically than the Protozoa: 80% vs 3.3 % and 83.3% vs 65.3 % respectively.

Akhtar (2010) carried out a study at the medical ICU (MICU) of The Holy Family Hospital in Rawalpindi, Pakistan from May 2007 to April 2008. Bacteria and Candida spp. were isolated from 60.1% samples taken from supposedly infected patients admitted at the medical ICU. The most frequent site of infection was the respiratory tract followed by urinary tract. P. aeruginosa, K. pneumoniae and E. coli were the common isolate, followed by MRSA and S. aureus.

Bouzada et al. (2010) evaluation of the susceptibility patterns of the isolated bacteria to disinfectant solutions showed sensitivities to different products routinely used in hospitals in concentrations ranging from 0.125 to 1%. Sodium hypochlorite was the compound which showed the highest inhibition concentrations (0.125 - 1%) and the CNS were the less susceptible bacteria to this disinfectant. Gram-negative rods were less susceptible to quaternary ammonium and to the association per acetic acid/ hydrogen peroxide in concentrations varying from 0.125 to 0.5%. The antimicrobial

susceptibility patterns for Gram-positive bacteria, among CNS strains higher than 10% resistance levels were observed against penicillin, oxacillin, erythromycin, azithromycin, clindamycin, chloramfenicol and sulfamethoxazole-trimethoprim, whereas lower levels of resistance were observed for the other antimicrobials with the exception of vancomycin to which all bacteria were susceptible.

Fakoorziba et al. (2010) investigated microbial contaminants of cockroaches (126 B. germanica and 69 P. americana) collected from four buildings (three public training hospitals and one house) in central Tehran, Iran. The oldest and largest of the three hospitals sampled (built 80 years ago) appeared to be the most heavily infested with cockroaches and cockroaches from this hospital accounted for most (65.4%) of the isolates of medically important bacteria encountered during the study. No significant difference was found between the %age of P. americana and B. germanica carrying medically important bacteria (96.8% vs 93.6%). Twenty five different species of medically important bacteria were isolated and identified; of these 22 were Gram- negative. The genus of enteric bacteria most frequently isolated from both cockroach species, at all four collection sites was Klebsiella. The cockroaches from each hospital were much more likely to be found contaminated with medically important bacteria than those from the house. The hospital collections of cockroaches also carried more medically important bacteria internally than externally (84.3% vs 64.1%).

Kaehn (2010) reported mechanism of action of PHMB as cytoplasmic membrane as the primary site of action resulting in modification of membrane permeability. PHMB binds with negatively charged phosphate head groups of phospholipids at bacterial cell wall resulting in increased rigidity, converting non-polar segments into hydrophobic domains, disrupting the membrane with subsequent cytoplasmic shedding and culminating in cell death. Electrostatic interaction of the PHMB with the acid phospholipids in the cytoplasmic membrane resulted in death of bacterial cell. PHMB effectiveness against bacterial cell is due to the biguanide group attached to a flexible spacer, a hexamethylene group. Maximal biocidal efficiency is obtained when six methylene groups are used as spacer between biguanide groups.

Kaleem et al. (2010) investigated in vitro susceptibility of MRSA against various antimicrobials. Vancomycin and linezolid were highly effective against MRSA.

Chloramphenicol and Minocycline also had good in vitro efficacy. Tetracyclin, though effective, enhances health care costs enormously, while 100% of the isolates were sensitive to linezolid.

Blazar et al. (2011) reviewed 11 different species of insects, including the Alphitobius diaperinus (A. diaperinus) (Panzer); Cochliomyia macellaria (C. macellaria) (Fabricius); Sarcophaga carnaria (S. carnaria) (L.); Musca domestica (M. domestica) (L.); Drosophila melanogaster (D. melanogaster) (Meigen); Stomoxys calcitrans (S. calcitrans) (L.)], P. americana (L.); B. germanica (L.); B. orientalis (L.); Diploptera punctata (D. punctata) (Eschscholtz); and N. cinerea (Olivier), which act as vectors for Salmonella spp. and E. coli and illustrated role of these insects as successful vectors of foodborne disease outbreaks.

Zafar et al. (2011) described prevalence of methicillin resistant Staphylococcus aureus (MRSA) outbreaks. Methicillin was introduced in 1960 as the first beta lactamase- resistant penicillin and first case of MRSA was reported in 1961. However outbreaks of MRSA infections were reported in Europe soon thereafter. Healthcare- associated MRSA were a major cause of nosocomial infections worldwide, with significant attributable morbidity and mortality in addition to pronounced healthcare costs. MRSA is also a major nosocomial pathogen in Pakistan and is emerging in the community. The overall prevalence of MRSA among S. aureus was documented as 42% in Pakistan though varying widely from 2–61%.

Adeleke et al. (2012) investigated the microbial load and antibiotic susceptibility pattern of pathogenic bacteria isolated from the faeces and body surfaces of cockroaches in Osogbo, Southwestern Nigeria. The microbial load of the microorganisms was significantly higher in the isolates from hospital as compared with the residential area (p<0.05) with the exception of Canidida species, Mucor and Penicillium which had higher or equal microbial load in residential areas. All the pathogenic bacteria isolated had multiple resistance to antibiotics most importantly, ampicillin, augumentin, amoxicillin and septrin (30 μg).

Akinjogunla et al. (2012) carried out a study in Uyo, Akwa Ibom State of South- Southern Nigeria. The bacteria isolated were Salmonella spp., Shigella spp., S.

aureus, coagulase negative Staphylococcus spp., B. cereus, E. coli, P. aeruginosa, K. pneumoniae, C. freundii, Morganella morganii (M. morganii), P. vulgaris, P. mirabilis, E. cloacae and Providencia spp. Among the gram-positive bacteria, only 75.8% S. aureus and 76.5% B. cereus were sensitive to streptomycin and gentamycin, while their resistance profiles to antibiotics in decreasing order was as follows: chloramphenicol (41.7%), amoxicillin (40.3%), streptomycin (36.1%), tetracycline (36.0%), erythromycin (35.5%), gentamicin (34.0%), penicillin (34.6%), cephalothin (27.8%), sulfamethoxazole (23.4%), ciprofloxacin (18.4%) and levofloxacin (17.7%). Less than 50% of E. cloacae and Providencia spp. were resistant to streptomycin, while ˂ 40% of P. vulgaris, K. pneumoniae, and P. aeruginosa were resistant to chloramphenicol. Of the 353 (67.8%) multidrug resistant bacteria, 121 (23.2%) were resistant to 3 antibiotics, 232 (65.7%) were resistant to 4-10 antibiotics. The antibiotic resistant Salmonella spp. and P. mirabilis had Multiple Antibiotic Resistance (MAR) indexes ranging from 0.27 to 0.82.

Bala and Sule (2012) conducted a study in Sokoto, Nigeria, and isolated a trematode S. haematobium eggs from cockroaches. Furthermore, isolation of coccidian parasites in Benin, indicated cockroaches having contact with fecal droppings of birds, dogs, and cats. Poor fecal and garbage disposal systems observed in Benin City may have contributed in a direct way to the parasitic contamination of these cockroaches.

Poly hexamethyl biguanide (PHMB) an environment friendly biocide was a new generation disinfectant with a wide scope of applications in all aspects of life. PHMB had broad spectrum of activity against bacteria, viruses and fungi. PHMB was used as swimming pool sanitizers and in cosmetics, leather preservatives, contact lens disinfectants, cleanser in agriculture and food handling, in treatment of hatching eggs, fibers and textiles and technical fluids like cutting oils and glues. PHMB was used commercially in antimicrobial hand washes and rubs and air filter treatments as an alternative to ozone (Bucher, 2012).

Feizhaddad et al. (2012) reported the pathogenic bacteria carried on the body surface of P. americana (L.) collected from three hospitals of Iran. All cockroaches were positive for at least three bacteria with the most common being E. coli (86.7%), followed by P. vulgaris (73.3%). While, the least recorded bacteria in all the analyzed

samples were B. cereus (66.7%), S. faecalis (60%), S. aureus (60%), E. cloacae (53.3%), Shigella (33.3%), Serratia (13.3%) and S. epidermidis (6.7%). On the contrary, cockroaches collected from Sina hospital, Iran, out of 3 hospitals carried all 9 bacterial species.

A descriptive and analytical study had been performed in five public hospital of Hamedan city, Iran by Jalil et al. (2012). P. americana, B. germanica, B. orientalis and P. australasiae were identified whereas, the most predominant species was B. germanica (88.8%) and the least common was B. orientalis (0.8%). Maximum number was collected from cook houses (32.4%) and the minimum from surgery ward (1.2%). The most predominant species of bacteria was E. coli (26.4%) and the lowest was Edwardsiella (0.4%). Frequency of bacteria isolated from external body surface and gut tract was not significantly different between P. americana and B. germanica.

Tilahun et al. (2012) conducted a five month study in neonatal intensive care unit of Tikur Anbessa Hospital, Ethiopia, and found no significant difference in the isolation of Citrobacter spp. and Klebsiella spp. from external surface and gut homogenates. The most numerous bacterial isolates were Klebsiella spp., Enterobacter spp., and Pseudomonas spp. also.

Alzain (2013) determined the possible role of cockroaches in dissemination of medically important gastrointestinal parasites in North Gaza Governorate, Palestine and reported that 23.5% of the cockroaches harbored parasitic organisms; 17.3% of the 110 cockroaches collected from residential areas harboured gastrointestinal parasites while 31.1% of the 90 cockroaches from hospital environment harboured parasites. Of these, 57.4% were Protozoa and the remaining 42.6% were pathogenic helminthes and non-pathogenic helminthes. Of the pathogenic helminthes, the species included ova of A. lumbricoides, ova of E. vermicularis and ova of hookworm. The Protozoa types that were identified included cysts of E. histolytica, trophozoites of B. coli, cysts of E. coli and cysts of E. nana isolated from internal and external surface of the cockroaches. The cockroaches from hospital environment harbored more parasites (31.1%) than residential areas (17.3%) but the difference in parasite burden was not statistically significant.

Al-Jailawi et al. (2015) observed that, when the concentration of Claradone is increased the growth of bacteria decreased. Growth of P. aeruginosa was slight when treated with 25% of Claradone, while moderate growth and good growth of bacteria was noticed when treated with 20% and 15%, 10% and 5% of Claradone, respectively. A number of survival colonies after treated with high concentration of Calarodone and Sarttol were investigated for their susceptibility to antibiotics, using standard disc diffusion method. These colonies of P. aeruginosa resisted the antibiotics that they were sensitive to before treatment. Thus, exposure to Claradone and sarttol can make the P. aeruginosa resistant against some antibiotics.

Chitsazi et al. (2013) reported isolation of Penicillium spp. (50%) in autumn and in spring Mucor spp. (57.14%) from German cockroaches collected from Mashhad Imam-Reza hospital, Iran, during 2009-2010. Cockroaches were also contaminated by 4 gram-positive bacteria species, including S. aureus, Enterococcus spp., Enterococcus spp. and Streptococcus spp.

Etim et al. (2013) carried out a study between January and June, 2011 in Anantigha area of Calabar, Nigeria and reported that 58.6% cockroaches were infected with one or several species of gastrointestinal parasites. Identified parasites included B. coli (8.8%), hookworms (9.6%), E. coli (10.4%), E. vermicularis (12. 9%), E. histolytica (13.7%), T. trichuira (16.9%) and A. lumbricoides (24.4%). Cockroaches collected from the toilets had the highest parasite load of 4-54 parasites/ ml followed by those from the kitchen with 1-24 parasites/ ml, and those from the living room 1-12 parasites/ml while 1-10 parasites/ ml was observed from cockroaches from the bedroom. More parasites were recovered from the external than in the gastrointestinal tract with prevalence amounting to 65.3 and 34.6%, respectively.

Hughes et al. (2013) proved role of disinfectants in reducing the infection rate of Clostridium difficile (C. difficile).the authors suggested that adopting various control measures including an improvement in surface disinfection of hospitals was the most likely way to reduce C. difficile infection rates. Weber et al. (2013) described the role of inanimate surfaces in the transmission of C. difficile and use of surface disinfection avoided transmission of C. difficile as uncontroversial.

Mikulak et al. (2013) assessed the role of the synanthropic cockroaches as the possible source of infections in the hospital environment in Poland in the years 2003- 2006. Several strains of bacteria and fungi were isolated from body of cockroaches e.g. E. cloacae, K. pneumoniae, C. freundii, S. marcescens, P. aeruginosa, S. epidermidis, Enterococus faecalis (E. faecalis), Enterococcus faecium (E. faecium), Trichosporon beigelii (T. beigelii), Fusarium moniliformei (F. moniliformes) and Scopulariopsis brevicaulis (S. brevicaulis). Many strains showed resistance to antibiotics and disinfectants. Several of isolated strains demonstrated important mechanisms of antimicrobial resistance likewise gram-negative bacilli - AmpC (+), ESBL (+), some strains of gram-positive cocci - MLSb and MRCNS.

Pai (2013) surveyed 69 long-term care facilities and nursing homes in Kaohsiung City, Taiwan, and found 45 (65.2%) sites were infested with cockroaches. Significant difference was observed for B. germanica trapped indoors (427 or 92.3%) than those trapped outdoor (12 or 2.7%) (P < 0.001) whereas no significant difference was observed between P. americana trapped indoors (63 or 52.9%) and outdoors (56 or 47.1%). Indoor environmental sanitation had significant association with cockroach infestation. Among P. americana and B. germanica, highest prevalence of bacterial isolate was Serratia spp. followed by Enterobacter spp. and Klebsiella spp. Gram- negative bacteria isolated from cockroaches were found to be resistant to ampicillin (10µg) and cephalothin (30µg). P. aeruginosa was the second frequently isolated glucose non-fermenter bacilli showing resistance to imipenem, ceftazidime, and cefepime whereas Acinetobacter spp. was not frequently found but exhibited resistance only to imipenem. Moreover, gram-positive bacteria revealed 100% resistance to oxacillin (1 µg) and pipemidic acid (20 µg).

Shahraki et al. (2013) studied cockroach infestation in urban communities of Yasuj City in southwestern Iran. Five cockroach species were identified including B. orientalis, B. lateralis, P. americana, B. germanica and S. longipalpa. Among these B. germanica was widely distributed (80% of infested sampling units) showing the highest frequency (96.7%) of trap counts. Comparison of rate and total infestation report via trapping (39%) showed that 33% reduction in cockroach infestation was due to regular cleaning of houses. %age frequency of the B. germanica in the five

types of study locations showed high abundance in the dormitories (98%), housing (95%), and hospital (93%) as compared to the other species.

Zacharia et al. (2013) investigated possible role of P. americana in carrying and transmitting human pathogens in India. Bacteria recovered from intestinal contents were Enterococcus spp. (95.6%), Klebsiella spp. (39.6%) and Proteus spp. (36%). Cockroaches were harboring Listeria spp. (51.2%) exhibited epidemiological significance in spread of nosocomial infection.

Alfa et al. (2000) revealed importance of surface disinfectants in controlling MRSA and other nosocomial disease agents. Significant decrease in MRSA, Vancomycin- resistant enterococci (VRE) and C. difficile infection rates was observed from ordinary cleaning of frequently touched surfaces in all disinfected patient rooms of the hospitals. The compliance in daily disinfection was monitored revealing significant effect on prevalence of MRSA and C. difficile.

Brown and Al Hassan (2014) investigated and compared multiple-antibiotic resistant bacteria carried by cockroaches from hostel and hospital environments. Both hospital kitchen and hostel cockroaches showed a potential of carrying similar bacteria, probably because both sampling areas were food preparation areas. Cockroaches trapped from the main kitchen of the hospital and the hostel carried multiple- antibiotic-resistant bacteria on their external or internal surfaces. Three gram-positive bacterial isolates being resistant to at least 4 of the 8 antibiotics tested however all were resistant to ampicillin, erythromycin and tetracycline whereas, 60% of the gram- negative enteric bacilli isolates were found resistant to at least 4 of the 8 antibiotics tested rather 90% enteric bacilli were susceptible to gentamicin and amikacin.

Brown and Al Hassan (2014) studied antibiotic resistance for bacterial isolates of cockroaches collected from kitchens of hospital and hostel. Cockroaches probably played an important role in spreading multiple-antibiotic-resistant Pseudomonas in both the hospital and surrounding buildings that was evidenced by 90% of P. aeruginosa isolates being resistant to 4 antibiotics. However, all P. aeruginosa were susceptible to amikacin and ciprofloxacin but resistant to ampicillin and tetracycline.

Since kitchen of the hospital was in close proximity to the wards, cockroaches perhaps easily accessed the wards and other infectious areas of the hospital.

Ganavadiya et al. (2014) compared efficacy of three chemical disinfectants (2% glutaraldehyde, 6% H2O2 and 99.9% ethyl alcohol) with distilled water as a control. The disinfecting efficacy was assessed on the basis of reduction in the total viable count. A statistically significant reduction in the total viable count was observed with glutaraldehyde, H2O2 and ethyl alcohol but not with distilled water.

Hamu et al. (2014) evaluated the role of B. germanica in mechanical transmission of intestinal parasites in Jimma town, southwestern Ethiopia. Overall, 152 (75.6%) of the 210 batches were found to harbor at least one species of human intestinal parasite. A. lumbricoides, T. trichiura, Taenia spp., Strongyloides like parasite, E. histolytica, Giardia duodenalis (G. duodenalis) and B. coli were identified from gut contents. Moreover, parasites were also isolated from the external surface in 22 (10.95%) cockroaches. Significant difference was observed in parasite carriage rate of the cockroaches among the study sites. Therefore, B. germanica was ascertained to harbor intestinal parasites of public health importance.

Issac et al. (2014) carried out a study in urban (Benin), Semi-urban (Ekpoma), and rural (Emuhi) areas in Edo State, Nigeria, between February and July 2013. Bacillus spp. and E. coli were the most common bacteria in cockroaches at all sites. However, E. faecalis was not found in cockroaches trapped from Ekpoma and Emuhi. A. niger was the most prevalent fungus in Benin and Ekpoma, while Mucor sp. was predominant in Emuhi. Parasitological investigations revealed the preponderance of A. lumbricoides in Benin and Emuhi, while T. trichura was the most predominant in Ekpoma. Seven fungi were isolated in Benin whereas 6 each were found for Ekpoma and Emuhi. Rhizopus spp. was seen only in cockroaches collected from Benin.

Kassiri et al. (2014a) examined American cockroaches from human dwelling localities of Ahvaz province, Iran, during 2008 and 2009 for the presence of bacteria on their external surfaces using specific standard methods for bacterial infection. Almost all the cockroaches in the residential environment carry medically important pathogenic bacteria. Eleven bacterial species were isolated from cockroaches. The

most common detected bacteria were found to be E. coli, S. aureus, Proteus spp., Klebsiella spp., Citrobacter spp. and Enterobacter spp. where 100%, 72%, 60%, 60%, 56% and 52% cockroaches were infected with these species, respectively. The minimum contamination was by Serratia spp. (20%), Micrococcus spp. (32%) and Enterococcus spp. (40%), respectively. The other extracted bacteria in this research were Psudomonas spp. (44%) and Sterptococcus spp. (48%).

Kassiri et al. (2014b) investigated contamination of American cockroaches with various bacteria in Khorramshahr Vali-e-Asr hospital, Iran, in 2008. Out of 20 collected cockroaches 18 (90%) were infected with at least 1 bacterium while 2 cockroaches (10%) were not infected. Various bacteria such as, Klebsiella spp. (35%), Pseudomonas spp. (30%), E. coli (15%), Staphylococcus spp. (10%), Proteus spp. (5%) and Streptococcus spp. (5%) were isolated. Klebsiella spp. was the most prevalent while Proteus spp. and Streptococcus spp. were the least prevalent bacteria recovered from external surfaces of cockroaches.

Masood et al. (2014) carried out a study on microbial screening of cockroaches in Quetta, Pakistan. Maximum number of infested American cockroach was collected from washrooms while minimum number was captured from kitchen. In contrast the maximum number of German cockroaches was captured from kitchen whereas few garden cockroaches were infested as well. From the total of 50 collected cockroaches, 32 (64%) showed bacterial load of E. coli and 41 (82%) showed a bacterial load of Salmonella.

Menasria et al. (2014) isolated and identified bacterial flora from B. germanica collected from dwellings and health facilities in Tebessa city, Tebassa Algeria. Three different bacterial species: Enterobacteria, Staphylococci and Pseudomonads group were recovered. The dominant S. aureus was followed by non-pathogenic staphylococci. Female cockroaches were found to be a high reservoir of S. aureus as compared to male cockroaches for both external and internal surfaces. Among the 187 bacteria isolated from collected cockroaches, 109 (RA=54.54%) belonged to the group of gram-negative Enterobacteria, and only 42 strains of Pseudomonas spp. were identified. P. aeruginasa (4.81%) presented 1 of the minor groups of the total isolated bacteria.

Menasria et al. (2014) tested antibiotic susceptibility patterns of S. aureus and P. aeruginosa isolates form B. germanica collected from dwellings and health facilities in Tebessa city, Tebassa, Algeria. All the isolated strains of P. aeruginosa were resistant to most tested antibiotics including: oxacillin, ampicillin, cefuroxime, pristinamycin, fusidic acid, erythromycin, vancomymcine and spiramycin. Whereas, only two antibiotics (rifampicin and clindamycin) showed anti-Pseudomonas activity with significant differences. All S. aureus isolates were found to be highly resistant to oxacillin (62.5%), ampicillin (75%), fusidic acid (87.5%) and erythromycin (75%). Susceptibility patterns of S. aureus isolates showed that majority of resistance was exhibited toward the commonly used antimicrobial.

Motevali Haghi et al. (2014) reported 100% yeast and 84.2% mold isolation from external surface of trapped cockroaches. No significant difference was found among the B. germanica and P. americana carrying medically important fungi. Candida spp. (94.7%) was the most prevalent yeast on external surfaces of cockroaches, followed by Rhodotrula spp. (57.9%). Whereas, Aspergillus spp. (84.2%), Fusarium spp. (15.8%), Penicillium spp. (10.6%) and Geotrichum spp. (10.6%) were the most prevalent molds appeared on external surfaces of cockroaches. C. glabrata (52.8%) and C. albicans (38.8%) were the most prevalent yeast, while A. niger (50%) was the most prevalent mold species isolated from cockroaches.

Okafor-Elenwo and Elenwo (2014) examined cockroaches for parasitic worms in Odau, a community in the Niger Delta region of Nigeria. The identified helminths were T. trichuria, A. duodenale, A. lumbricoides, S. stercoralis, and Trichostrongylous spp.: 79.1% of the total cockroaches had parasitic helminths. The rate of occurrence of Ascaris spp. in the cockroaches was significantly higher (p<0.05) compared to the other parasites. The abundance of helminth parasitic species in the cockroaches indicated poor sanitary condition.

Okore et al. (2014) found Dettol as the most effective on E. coli (6.5%) followed by Jik (4.5%) then Izal and Z-germicide (4%). Against S. aureus Dettol was the most effective (6.25), followed by Zgermicide (4), both Izal and Jik were less effective on S. aureus than pheol. Dettol is the most effective on Streptococcus spp. (6.25%), followed by Izal (4%), but Jik and Z-germicide are less effective on Streptococcus

spp. The different dilutions of the disinfectants gave different zones of inhibition in the range of 3.5mm to 11.0 mm (Jik). The antimicrobial activity of Dettol was more on the gram-positives Streptococcus spp. (30mm) and S. aureus (28mm).

Vazirianzadeh et al. (2014) conducted research on 39 Brown-banded cockroaches collected from kitchen area of houses, Ahvaz SW, Iran and recovered 179 bacterial agents were isolated. Among the isolated microbes, 92 were from alimentary ducts and 87 from external body surfaces. These isolated bacteria from cockroaches were identified as Enterobacter spp., Klebsiella spp., Citrobacter spp., E. coli, Salmonella spp., Proteus spp., coagulase negative staphylococci, S. marcescens, S. aureus, and Bacillus spp. The resistance patterns were determined for gram-negative bacilli and gram positive cocci against 18 antibiotics. Among all the gram-negative bacilli isolates resistance rates were above 52.4% for ampicillin, cephalothin, ceftazidime, cefalexin and tetracycline while cefotaxime expressed the highest susceptibility among all the isolates from the kitchen area of houses. Among gram-positive cocci isolates resistance rates were above 53.8% for ampicillin, amikacin, penicillin, ceftazidime, nitrofurantoin, nalidixic acid, trimethoprim- sulfamethoxazole, cefalexin, cefotaxime and tetracycline, while ciproflexoxacin expressed the highest susceptibility among all the isolates from the kitchen area of houses.

Wannigama et al. (2014) investigated prevalence and antibiotic resistance of gram- negative pathogenic bacteria species isolated from P. americana and B. germanica in Varanasi, India. No significant difference was observed between the overall bacterial load of the external surface in P. americana (64.04%) and B. germanica (35.96%). The predominant bacteria on cockroaches were K. pneumonia, E. coli, E. aerogenes, and P. aeruginosa; however, K. pneumoniae and P. aeruginosa were the most prevalent, drug-resistant strains were isolated from the cockroaches with 100% resistance to sulfamethoxazole/ trimethoprim and ampicillin. While evaluating MDR for individual strains of bacterial isolates, E. coli and C. freundii were resistant to four antibiotics tested, and E. aerogenes and P. mirabilis to three antibiotics tested.

Akbari et al. (2015) identified the bacterial microbiota of midgut of P. americana. The analytical profile index (API) kit identified 11 bacterial species including: E. coli, S. flexineri, C. freundii, E. vulneris, E. cloacae, Yersinia pseudotuberculosis (Y.

pseudotuberculosis), Y. intermedia, Leclericia adecarboxylata (L. adecarboxylata), K. oxytoca, K. planticola, and Rahnella aquatilis in the cockroach midguts. E. coli, S. flexineri, and C. freundii are potentially symbiotic whereas other isolates are transient or temporary. The symbiotic isolates were present in the midgut after washing while the transient bacteria were washed out in normal saline. The anterior colon of the cockroaches contained the highest abundance of microorganisms. The diversity in different compartments of cockroach gut was related to the microbial activities, such as the accumulation of hydrogen and other microbial products, and the physiochemical characteristics of each part of the gut, such as pH and redox potential. The midgut of P. americana had an endodermic origin that was not destroyed in molting and its microbial community remained intact during multiple molting of cockroach life span.

Ghasemi-Dehkordi et al. (2015) carried out study in Chaharmahal Va Bakhtiari province (southwest Iran) and reported detection of E. coli infection in 84 samples (76.36%) and 11 samples had the TEM-1 gene (10%). The antibiotic susceptibility pattern using the disk diffusion test indicated that most E. coli isolates from cockroaches were sensitive to imipenem (100%) and amikacin (87.8%), while resistance to aztreonam, ceftazidime, and ceftriaxone was 88.1%, 80.5%, and 78.2%, respectively. The increasing antibiotic resistance of bacteria like ESBLs in E. coli pathogenicity is important and resistance genes such as TEM-1 are risk factors for transmission of gastrointestinal infections to humans.

Hizbullah et al. (2015) investigated the recent trends in the prevalence and antibiotic susceptibility of MRSA. Four hundred and fifty clinical samples were assembled at National Institute of Health, (NIH), US, from January to March 2015. The prevalence of MRSA was found higher in pus 51.56%, followed by blood 28.17%, urine 18.43% and sputum 8.12%. MRSA positive samples were collected mainly from surgical wards followed by medical wards, Paediatrics wards and outpatients. All isolates were found sensitive to vancomycin and teicoplanin, while sensitivity to Novobiocin 80.31% clindamycin was (53.12%), chloramphenicol (45%) and ciprofloxacin was 25%.

Iboh et al. (2015a) conducted a study in Yakurr community of Cross River State, Nigeria and identified micro-organisms from the external body surfaces of cockroaches trapped from different houses with open toilets and with broken sewage systems. A total of 352 cockroaches were caught from both sites; of these 331 (94.0%) were positive for bacteria and parasites. The bacterial species identified were K. pneumonia, E. cloacae, E. aerogenes, Salmonella spp., Shigella sonnei, V. cholerae, and C. freundii. Parasitic contaminants included B. coli trophozoites, E. histolytica (cysts), A. lumbricoides (ova), T. trichura (ova), E. vermicularis (larvae), A. duodenale (larvae) and S. stercoralis (larvae). No significant difference in the parasitic and bacterial infection rates of cockroaches was observed irrespective of the sampling site.

Iboh et al. (2015b) investigated the different parasites harbored and transmitted by cockroaches within households in Calabar Municipality, Nigeria. A total of 348 parasites were recovered and identified as A. duodenales larvae, A. lumbricoides ova, E. vermicularis ova and larvae, S. stercoralis ova and larvae, E. histolytica cysts and Fusarium spp.

Jabber et al. (2015) investigated the bacterial load and antibiotic susceptibility pattern of pathogenic bacteria isolated from the guts and body surfaces of cockroaches in Haboubi Hospital, the province of Dhi Qar, Iraq. The distribution of the isolated bacteria included the highest values of Salmonella spp. and P. mirabilis, and the lowest values of Aeromonas hydrophila (A. hydrophila) and Raoultella ornithinolyticae (R. ornithinolyticae). The microbial load was higher in the outer surface (94%) as compared to guts (6%). Gram-negative bacterial loads isolated from cockroaches reached the highest for S. marcescens (11.6%), followed by P. mirabilis (11.2%), and much lesser loads of A. hydrophila (0.7%) and R. ornithinolyticae (1.1%).

Meyer et al. (2015) reported role of disinfectants in controlling rate of bacterial infection in an outbreak and recommended that surface disinfection must be done correctly in order to reduce the rate of infection. An appropriate efficacy spectrum of the chosen disinfectant revealed surfaces were completely wet with sufficient amount of liquid and a mechanical wiping of the surfaces was vital.

Yaro et al. (2015) determined the role of cockroaches as potential carrier of S. stercoralis in Ahmadu Bello University Main Campus and Samaru Village, Zaria, Kaduna State, Nigeria. S. stercoralis were carried by cockroaches captured in Zaria at a prevalence of 8.57% with a prevalence of 9.26% in Ahmadu Bello University (ABU) and 7.54% in Samaru Village. Cockroaches were observed to carry S. stercoralis significantly more on the external body surface (7.46%) than on the internal body surface (1.90%) and in nymphs (44.14%) than adults (4.24%). The mean load of S. stercoralis was highest in Queen Amina Hall (3.65) and least in Suleiman Hall in ABU (0.13). No S. stercoralis was recovered from cockroaches from Akenzua Hall, ABU and Danraka Area, Samaru Village. Thus cockroaches proved as reservoir for S. stercoralis, the causative agent of strongyloidiasis in human habitat.

Kogi et al. (2016) determined the role of cockroaches as carriers of Moniliformis dubius (M. dubius) in Ahmadu Bello University (ABU) Main Campus, Zaria, Kaduna State, Nigeria. Only P. americana harboured this parasite while B. germanica harbored no M. dubius. M. dubius isolated from male and female P. americana revealed no significant difference (P>0.05) with the female and male having a prevalence of 12.50% and 12.08% respectively. M. dubius isolated from adults. P. americana (20.83%) was highly significant from the nymphs (3.75%). M. dubius obtained from the body surfaces of P. americana showed that the parasite dominated more in the internal body surface (10.58%) than the external body surface (7.14%), but these values were statistically not different.

Moges et al. (2016) assessed the bacterial isolates and their antimicrobial profiles from cockroaches in Gondar town, Ethiopia. Of the 181 identified bacteria species, 110 (60.8%) were identified from external and 71 (39.2%) from internal parts of cockroaches. K. pneumoniae 32 (17.7%), E. coli 29 (16%), and Citrobacter spp. 27 (15%) were the predominant isolates. High resistance was observed to cotrimoxazole, 60 (33.1%), and least resistance was noted to ciprofloxacin, 2 (1.1%). Additionally, 116 (64.1%) of the isolates were MDR strains; Salmonella spp. were the leading MDR isolates (100%), followed by Enterobacter (90.5%) and Shigella spp. (76.9%).

Morenikeji et al. (2016) determined the prevalence of parasites in cockroaches recovered from residential houses around Awotan dumpsite in Ido Local Government Area of Oyo state, Nigeria. Parasites recovered and identified included S. stercoralis (80.6%), N. ovalis (7.9%), H. diesingi (2.9%), Toxascaris leonina (T. leonine) (1.4%), E. vermicularis (0.7%) and a fluke (4.3%). More parasites (83.5%) were recovered from the gut than from the external body part (16.5%) of the infected cockroaches. Residents around the dump sites were prone to parasitic infestation through mechanical transmission of parasites by cockroaches.

Sia Su et al. (2016) conducted a study in three randomly selected areas of Metro Manila, namely Manila, Pasay, and Quezon City, Phillipines. The common parasite observed in the sampled cockroaches was the rhabditiform larva (25%). Significant difference was observed between parasites isolated from the cockroaches collected from the selected areas. Higher parasite community diversity was observed in cockroaches collected from Manila, whereas lower parasite community diversity was observed in Quezon City though the abundance of parasites was seen on the cockroaches.

Solomon et al. (2016) determined the vector potential of cockroach for medically important bacterial pathogens in restaurants and cafeterias. Cross-sectional study was conducted on cockroaches from restaurants and cafeterias in Jimma town, Ethiopia, from May to September 2014. E. coli was the most frequently isolated followed by Salmonella spp., B. cereus, and S. flexneri. Klebsiella spp. 49(40.8%), Bacillus spp. and S. saprophyticus were predominant bacterial isolates from P. americana. B. germanica (L.) served as a potential vector for the dissemination of foodborne pathogens such as Salmonella spp., S. flexneri, E. coli, S. aureus and B. cereus and were major threat to public health.

Despite the lack of definite direct evidence that cockroaches are vectors of bacterial agents causing human diseases, the potential of health risks associated with the ability of these insects to carry multiple antibiotic-resistant bacteria, necessitates control measures of cockroaches to be taken in hospitals, restaurants and houses to avoid direct or indirect human contact with them or with their digestive tract discharges.

MATERIALS AND METHODS

3.1: Experimental sites

The research was conducted at the:- i) Entomology Research Laboratory, Department of Zoology, Lahore College for Women University, Lahore, Pakistan. 3.2: Study area The study area included the urban locations of Lahore District spread over 1772 km2. According to the 2013 population census of Pakistan, population of District Lahore stood at 12,218,345; of which 81.17% was urban and this urban component of population is increasing day by day. Lahore lies between 31°15′—31°45′ N and 74°01′—74°39′E at an elevation of approximately 216 m above sea level. Lahore features 5 seasons semi-arid climate; i.e. foggy winters (15 Nov - 15 Feb) with few western disturbances causing rains, pleasant spring (16 Feb - 15 April), summer (15 April – 30 June) with dust rain storms and heat wave periods, rainy monsoon (1 July - 16 September) and dry but pleasant autumn (16 September-14 November) (Punjab Meteorology Department, 2014). The study was conducted in different hospitals, shopping malls/stores, institutes and urban residential areas of the city which are densely populated.

3.3: Collection and identification of cockroaches

3.3.1: Collection of cockroaches

In the present study, cockroach species composition, abundance, diversity and distributional patterns were determined in the study area in four different seasons around one year. Cockroaches were observed and collected randomly from April 2013 to March 2014 with the help of sticky traps, food-baited pitfall traps and manual catching by hand. Specimens were collected from 20 different sites including hospitals (e.g. Punjab Institute of Cardiology, Mayo Hospital, Sheikh Zaid Hospital, Jinnah Hospital and General Hospital) shopping malls/stores (e.g. Swera Departmental Store, Metro Cash and Carry and Hyperstar), institutes/office (e.g. LCWU, Punjab University, UVAS, GCU Lahore and PASSCO), houses (e.g. Mughalpura, Model Town, Shadman Colony, Shalamar Town, Maraghzaar Colony, Johar Town and Jallo Town) (Fig. 3.3.1).

Geo-coordinates of each study site by using Global Positioning System (GPS) receiver are given in Table 3.3.1. Traps were kept on the floor close to the wall of rooms, under cupboards, beds, storage racks, under washbasins and pantries. Each trap was placed in living room, bedroom, bathroom and kitchen of houses, different wards, store rooms, kitchen stores, canteen area of hospitals, in grocery areas, food retailing areas of shopping stores and in each working room of the institute/office units for three consecutive nights. Nymphs along with adults were heavily trapped in baits and traps.

Table 3.3.1: GPS location of cockroach collection sites in urban areas of Lahore, Pakistan

Sr. No. Collection sites Latitude Longitude 1 Punjab Institute of Cardiology 31°32'18.48"N =31.5384667 74°20'9.28"E =74.3359111 2 Mayo Hospital 31°34'18.06"N =31.5716833 74°18'57.04"E =74.3158444 3 Shaikh Zayed Hospital 31°30'29.82"N =31.5082833 74°18'30.17"E =74.3083806 4 Jinnah Hospital 31°29'3.93"N =31.484425 74°17'48.40"E =74.2967778 5 General Hospital 31°27'17.46"N =31.45485 74°21'0.94"E =74.3502611 6 Swera Departmental Store 31°25'54.81"=31.4318917 74°17'11.38" =74.2864944 7 Metro Cash & Carry 31°29'34.65"N =31.4929583 74°25'1.57"E =74.4171028 8 Hyperstar 31°32'5.69"N =31.5349139 74°21'47.79"E =74.363275 9 LCWU, Lahore 31°32'41.85"N =31.5449583 74°19'37.97"E =74.3272028 10 Punjab University, Lahore 31°29'44.24"N =31.4956222 74°17'39.17"E =74.2942139 11 UVAS, Lahore 31°34'29.03"N =31.5747306 74°17'57.48"E =74.2993 12 GCU, Lahore 31°34'22.14"N =31.5728167 74°18'29.22"E =74.3081167 13 Passco office 31°33'36.68"N =31.5601889 74°19'56.62"E =74.3323944 14 Mughalpura 31°33'46.53"N =31.562925 74°22'49.35"E =74.380375 15 Model Town 31°28'37.18"N =31.4769944 74°19'44.66"E =74.3290722 16 Shadman Colony 31°32'14.97"N = 31.5374917 74°19'50.76"E = 74.330767 17 Shalamar Town 31°35'12.47"N = 31.5867972 74°22'55.29"E = 74.382025 18 Maraghzaar Colony 31°29'44.24"N =31.553326 74°17'39.17"E =74.305122 19 Johar Town 31°27'43.38"N = 31.46205 74°17'38.90"E = 74.294139 20 Jallo Town 31°35'47.57"N = 31.5965472 74°29'57.97"E = 74.499436

Figure 3.3.1: Map of Lahore District showing the collection sites of cockroaches including hospitals and houses.

3.3.2: Identification of cockroaches

Field-collected specimens of cockroaches were transported to the Entomology Research Laboratory, Lahore College for Women University, Lahore, Pakistan for identification to species level. Cockroach sex was determined by presence/absence of male styli in addition to paired cerci on the 9th abdominal sternum. Species description for the genus Blattella was based on the morphology of adult male (Roth, 1985), therefore only adult male specimens were used to confirm their identification. Cockroach nymphs closely resembled their adults except that the nymphs were generally smaller and lacked wings and genital openings or copulatory appendages at the tip of their abdomen. Taxonomic identifications were made by observing morphological features already published in various pictorial and published keys (Pratt and Stojanovich, 1966; Abul Hab, 1980; Hagenbuch et al., 1988; Borror et al., 1989; Roth, 1995; Choate, 2009). Morphological study comprised of body length, body colour, presene/ absence of stripes on head shield and abdomen, winged or wingless, wing length, functional/nonfunctional wings, ootheca characterization, nymphal stages characteristics and number of molts. Species abundance and richness was evaluated in 4 trimesters extending from April, 2013 to March, 2014. First trimester covered April - June, 2013; 2nd trimester covered July - September, 2013; 3rd trimester covered October – December, 2013; while the last 4th trimester covered January – March, 2014.

3.3.3: Weather data collection

Average monthly minimum and maximum air temperature and relative humidity data were obtained from the Punjab Meteorological Department, Lahore, and were correlated with prevailing population density of cockroaches.

3.3.4: Data analysis

The observations were tabulated and data were statistically analyzed by using Microsoft excel. Relative abundance of each cockroach species was calculated. Species richness and evenness of each cockroach species was calculated for each sampled trimester. Diversity of different cockroach species on the Simpson and Shannon index was analyzed according to Simpson (1949) and Shannon-Wiener function (Shannon and Weaver, 1949) as follows:-

Relative abundance (Pi) = Number of individuals (spp.)/ Total population

= Ni/N

Species Richness (S) = Number of different species in a given area

Species evenness = Shanon Wiener Diversity index / Natural logarithm of species richness

= H′ / ln S

Shannon-Wiener diversity index (H′) = - [∑ Pi (ln Pi)]

Shannon-Wiener Diversity Index was calculated in order to know the species diversity in different habitats as the presence of one individual of a species is not necessarily indicative of that species being present in a large number. A greater number of species may share similar abundances within the community, with resulting in an increase in Shannon's diversity. Shannon-Weiner Index assumes that individuals are randomly sampled from an independently large population and all the species are represented in the sample. Shannon diversity is very widely used index for comparing diversity between various habitats (Clarke and Warwick, 2001). The maximum Shannon's diversity for a sample is found when all species are equally abundant. Values of the Shannon's diversity index for real communities typically fall between 1.5 and 3.5.

Diversity index (D) = Σ(Pi)2

Simpson Diversity index = 1-D

Simpson diversity index measures the probability that two individuals randomly selected from a sample were belonging to the same species. Simpson gave the probability of any two individuals drawn from noticeably large community belonging to different species (Simpson, 1949).

Average temperature of each trimester was compared with abundance of different cockroach species and mean and standard deviation values; probability was determined by one-way analysis of variance (ANOVA), Post-hoc comparisons using the Fisher Least Significant difference (LSD) test; Pearsons coefficient for correlation were determined besides descriptive statistics using the SPSS software (IBM SPSS Statistics 20). Significance was set at P< 0.05 level.

3.4: Isolation and identification of microbes from external surface of cockroaches

3.4.1: Sample collection

A set of randomly selected 160 cockroaches (50 from hospitals and 110 from houses) trapped during April – September, 2013 from different wards of 5 hospitals and from bed rooms, kitchens and store rooms of 11 houses in different towns in urban area of Lahore were selected for isolation and identification of microbes from their external surfaces. Each individual cockroach was conserved in a sterile bottle and transported to the laboratory for microbiological analysis.

3.4.2: Isolation of bacteria from external surfaces of cockroaches

The experimental cockroaches were immobilized by placing them in a freezer at -4°C for 5 min; 2 ml of sterile normal saline (0.9%) was added to a test tube containing 1 cockroach, and the tubes were shaken thoroughly for 2 min to isolate microorganisms from the external surface of the cockroach. The cockroach was then transferred to another sterile tube using forceps.

3.4.3: Identification of bacteria from external surfaces of cockroaches

3.4.3.1: Preparation of media

Media are substrates that support growth of the microbes. Various types of media i.e., selective media, enrichment media and differential media were prepared to identify different bacterial isolates.

3.4.3.1.1: Preparation of Tryptic Soy Agar (TSA)

40 gm of dehydrated TSA media (Lab M Limted, Cat # Lab 11) was suspended in 300 ml distilled water. Final volume was made upto 1 litre by adding distilled water. Media was sterilized at 121°C for 15 min then cooled to 45-50°C. Flask was gently shaken to mix media before dispensing into sterile petri dishes (Appendix I).

3.4.3.1.2: Preparation of Mannitol Salt Agar (MSA)

Mannitol Salt Agar is used as a selective medium for the isolation of pathogenic Staphylococci. 111 gm of Mannitol Salt Agar powder (Himedia, Cat # M118) was suspended in 300 ml of distilled water and mixed well. Final volume was made upto 1000 ml by adding distilled water. Media was boiled to dissolve it completely. Media

50 was sterilized at 121°C for 15 min than cooled to 45-50°C. Flask was gently shaken to mix media before dispensing into sterile petri dishes (Appendix II).

3.4.3.1.3: Preparation of MacConkey Agar

MacConkey Agar (Merck, Germany Cat # VM327265) with crystal violet, sodium chloride and 0.15% bile salts is a differential medium recommended for the selection and recovery of the Enterobacteriaceae and related enteric gram-negative bacilli. MacConkey Agar (51.55 gm) was suspended in 300 ml of distilled water. And after completly dissolving the media, the final volume was made upto 1000 ml by adding more distilled water. Agar was heated to boil with gentle swirling for dissolving it completely. Media was sterilized by autoclaving at 121°C for 15 min then cooled to 45-50°C. Flask was shaken gently to mix media before dispensing into sterile petri dishes (Appendix III).

3.4.3.1.4: Preparation of Eosin Methylene Blue Agar (EMB)

EMB Agar is recommended for the isolation and differentiation of gram negative enteric bacteria from clinical and nonclinical specimens. Methylene blue and Eosin-Y inhibit gram positive bacteria. 36 gm of EMB Agar (Lab M Limited, Cat # Lab61) was suspended in 300 ml of distilled water and final volume was made upto 1000 ml by adding more distilled water. The medium was heated to dissolve it completely. Media was sterilized by autoclaving at 121°C for 15 min than Cool to 45-50°C. Flask was shaked gently to oxidize the methylene blue and to suspend the flocculent precipitate before dispensing into sterile petri dishes (Appendix IV).

3.4.3.1.5: Preparation of Salmonella Shigella Agar (SS agar)

A differential, selective medium is recommended for the isolation of Salmonella and Shigella from stool, foods and clinical material. 63 gm SS agar (Bio-Rad France, Cat # 64514) was suspended in 300 ml of distilled water and final volume was made upto 1000 ml by adding more distilled water and then brought it to boiling with frequent agitation and simmered gently to dissolve the agar completely and then cooled to about 50°C and mixed well before dispensing into sterile petri dishes (Appendix V).

3.4.3.1.6: Preparation of Blood Agar Plate (BAP)

Blood agar is a non-selective medium for the isolation and cultivation of many pathogenic and non-pathogenic microorganisms like Neisseria, Streptococci, etc. The

51 medium is often used to observe the forms of haemolysis from pathogenic microorganisms. 40 g of BAP media (Sigma aldrich Cat # 70133) was suspended in 300 ml of distilled water and final volume was made upto 1000 ml by adding more distilled water and boiled to dissolve completely. The media was sterilized by autoclaving at 121°C for 15 min. For blood agar autoclaved media was cooled to 45- 50°C and aseptically 6% of sterile defibrinated blood was added before dispensing into sterile petri pates (Appendix VI).

3.4.4: Inoculation of sample

Aliquot (0.01 ml) of the saline washing of cockroaches were then separately inoculated onto the surface of selective and differential agar plates such as tryptic soy agar (TSA), mannitol salt agar (MSA), macConkey agar, eosine methylene blue agar (EMB), Salmonella Shigella agar (SSA) and blood agar plate (BAP). The agar plates were incubated at 37°C for 24 hrs.

3.4.5: Identification of microbes

The bacteria grown on the selective and differential agar media were identified by microscopic, colonial morphology, Gram staining, and biochemical tests such as oxidase, catalase, coagulase, indole, methyl red and voges proskaeur (MRVP) (Hensyl, 1994).

3.4.5.1: Microscopic colonial morphology

Pure cultures were isolated by streak plate method and pour plate method. The obtained pure colonies were identified by using standard bacteriological procedure as described in Burgey‘s Manual of Determenative Microbiology. The colony character of one bacterial species was different from those of other bacterial species. Different criteria for describing the colony characters were size, shape, edge, surface, color, consistency, elevation etc. Therefore colony appearance was a valuable clue to identify a bacterial culture and to confirm its purity (Cowan and Steel, 1974).

3.4.5.2: Gram Staining

A drop of double distilled H2O was placed on clean sterile glass slide and a loop of culture from a bacterial colony was transferred on slides and suspension was prepared. Suspension of the culture was spread on the slide and allowed for drying. Smear was fixed by gently passing the slide over the flame for 2 to 3 times with smear

52 surface on the top. The smear was flooded with basic stain crystal violet (Appendix XI) and the stain was allowed to remain on the slide for 1 min. After that, slide was washed in slow running water to remove extra stain. 3 drops of Gram Iodine solution (Appendix XII) were applied on smear for 1 minute and washed with water to remove extra Gram Iodine solution. Smear was decolorized with 95% ethanol (decolorizer) (Appendix XIII) for 15 sec and washed with water. 3 drops of Safranine (counterstain) (Appendix IV) were applied on smear for 1 min and then washed it with slow running water. Smear was allowed to dry in air and a drop of immersion oil was placed on smear to observe it under the oil immersion objective. Depending upon stain, the bacterial film was observed either violet color (Gram positive bacteria) or red color (Gram-negative bacteria).

3.4.5.3: Biochemical tests

3.4.5.3.1: Oxidase Test

The oxidase test is used to identify bacteria that produce cytochrome-c oxidase, an enzyme of the bacterial electron transport chain. A filter paper soaked with the substrate, Tetramethyl-p-Phenylenediamine Dihydrochloride was taken (Appendix XV). The paper was moistened with sterile distilled water. The bacterial colony to be tested was picked with wooden or platinum loop and smeared on the filter paper. Inoculated area of paper was observed for a color change. Deep blue or purple color appeared within 10-30 sec for positive samples.

3.4.5.3.2: Catalase test

Catalase is an enzyme, produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites, H2O2. The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme.

Small amount of bacterial colony was transferred to a surface of clean, dry glass slide using a loop or sterile wooden stick. 3% H2O2 was dropped on to the slide and mixed well (Appendix XVI). A positive result was the rapid evolution of oxygen (within 5- 10 sec.) as evidenced by bubbling. A negative result was no bubbles or only a few scattered bubbles.

3.4.5.3.3: Coagulase test

Coagulase test is used to differentiate S. aureus (positive) from Coagulase Negative Staphylococcus (CNS). Coagulase is an enzyme produced by S. aureus that converts soluble fibrinogen in plasma to insoluble fibrin.

3.4.5.3.3.1: Slide coagulase test

A staphylococcal colony was emulsified in a drop of water on a clean and grease free glass slide with a minimum of spreading. Similar suspensions of control positive and negative strains were made to confirm the proper reactivity of the plasma. A flamed and cooled straight inoculating wire was dipped into the undiluted plasma at room temperature, withdrawn, and stirred the adhering traces of plasma into the staphylococcal suspension on the slide (Appendix XVII). The wire was flamed and repeated for the control suspensions. A coarse clumping of cocci visible to the naked eye within 10 sec was read as positive and the absence of clumping or any reaction taking more than 10 sec to develop was considered as negative results.

3.4.5.3.3.2: Tube coagulase test

6 dilutions of the plasma in saline were prepared and 1 ml of the diluted plasma was placed in small tubes. Bacterial colonies were emulsified in 1 ml of diluted rabbit plasma to give a milky suspension (Appendix XVII). Tubes were incubated in incubator at 35oC for 4 hrs. Tubes were examined at regular intervals of 1, 2 and 4 hrs for clot formation by tilting the tube through 90º angle. The negative tubes were left at room temperature overnight for re-examination.

3.4.5.3.4: Indole test

Indole test was used to determine the ability of an organism to split amino acid tryptophan into a compound indole. Tryptophan is hydrolysed by tryptophanase to produce three possible end products – one of which is indole. Indole production is detected by Kovac‘s reagent which contains 4 (p)-dimethylamino benzaldehyde, this reacts with indole to produce a red coloured compound. Indole test helps to differentiate Enterobacteriaceae and other genera.

10 gm of Peptone (Riedel-deHaen Cat # 19657) was added in 100 ml of dd.H2O and dispensed and sterilized by autoclaving at 121°C for 15 min. Peptone water broth was inoculated with isolated colony of the test organism and emulsified and incubated at

37°C for 24-28 hrs in ambient air. 0.5 ml of 1% Kovac‘s reagent was added to the peptone water broth culture (Appendix XVIII). Pink colored ring appeared at top after addition of Kovac‘s reagent for positive samples.

3.4.5.3.5: Methyl Red-Voges Proskaeur (MRVP) Test

MR-VP broth (Buffered Peptone Glucose Broth) was used for the differentiation of bacteria by means of the methyl red and Voges-Proskauer reactions (Appendix XIX). 17 gm of buffered peptone glucose broth was dissolved in 300 ml distilled water and final volume was made upto 1 litre by adding distilled water and then mixed thoroughly and heated slightly to dissolve it completely. 5 ml broth was poured in each test tube and autoclaved at 121°C for 15 min, half the material in the test tube was used for MR test and the other half for VP test. Using a light inoculum, tubes of MR-VP media were inoculated with 18-24 hrs pure bacterial cultures. Tubes were incubated aerobically at 35±2°C for 4-5 days. After appropriate incubation period, test tubes were removed and subjected for further testing of MR and VP tests.

3.4.5.3.5.1: Methyl red Test

5 drops of methyl red indicator were added to test tubes of the buffered peptone glucose culture broth (Appendix XX). The change in color of the medium to red within a few minutes indicated the positive reaction.

Clark and Lubs, (1915), discovered the ability of coliforms to produce and maintain acid products when cultivated in specific media. Methyl Red (MR) test was used to determine whether the microbe performs mixed acids fermentation when supplied with glucose. In mixed acid fermentation, three acids (acetic, lactic and succinic) were formed in significant amounts. These large amounts of acids resulted in significant decrease in the pH (lower than 4.4 pH units) of the medium towards more acidic. It was visualized by using pH indicator, methyl red (p-dimethylaminoaeobenzene-O- carboxylic acid), which was yellow above pH 5.1 and red at pH 4.4.

3.4.5.3.5.2: Voges-Proskauer (VP) test

10 drops of Barritt's A reagent and 10 drops of Barritt's B reagent were added to inoculated buffered peptone glucose broth concurrently (Appendix XXI). The test tubes were shaken gently for few minutes. Formation of red color within 15 to 20 min indicated positive result, while no red color formation after 15 to 20 min was indicative of negative result.

Voges and Proskauer (1898) first observed the production of a red color after addition of potassium hydroxide to cultures grown on specific media. Certain bacteria produce neutral-reacting end products when cultivated in specific media. Particular enteric bacteria that ferment glucose further metabolize pyruvic acid to form acetyl-methyl carbinol (acetoin). This end product, in the presence of atmospheric oxygen and 40% potassium hydroxide is converted to diacetyl. Diacetyl, under the catalytic action of alpha-naphthol and creatine, is converted into a red complex. This is a positive Voges-Proskauer (VP) test reaction. The VP test is used primarily to separate E. coli (VP-negative) from the Klebsiella - Enterobacter groups (VP-positive).

3.4.5.3.6: Citrate test

Koser (1923) developed a liquid medium to differentiate E. coli and E. aerogenes. This medium contained a single nitrogen source supplied by an inorganic ammonium salt and a single carbon source supplied by sodium citrate. In 1926, Simmons modified Koser‘s formulation by adding 1.5% agar and bromothymol blue indicator. Citrate utlilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic ammonium dihydrogen phosphate is the sole fixed nitrogen source.

Simmons Citrate agar (Sigma Aldrich Cat # 85463) (24.2 g) was suspended in 1 litre of demineralized water (Appendix XXII). Media was heated to boiling with agitation to completely dissolve it. 10 ml media was dispensed into each test tube and sterilize by autoclaving at 121°C for 15 min and the test tubes were cooled in a slanted position for use as slants. The citrate slants were inoculated lightly in streaking way by pure bacterial colony. Slants were incubated at 35oC to 37oC for 24 hrs. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium. Development of intense Prussian blue color of slants denoted alkalinization and visible growth on the slant surface.

3.4.6: Statistical analysis of bacterial isolates

Bacterial infection rate for each site was calculated for both species of cockroaches in relation to external bacterial infection.

Bacterial infection rate = Total number of bacteria isolated X 100 Total infected cockroaches

Calculations using Shannon-Wiener‘s diversity index (H′) (Shannon and Wiener, 1949) were conducted to measure the bacterial community diversity (all species of bacteria) in each habitat:

H′ = −ΣPilnPi

Pi = ni/N

Where, Pi is the proportion of the specific bacterial species in the total sample, N is the number of species richness.

Jaccard‘s index of similarity was used to analyze the similarity of the bacterial community among habitats (Jaccard, 1912). p = c/ (a + b + c) where, ―p” is Jaccard‘s index of similarity, ―‖c is the number of the shared bacterial species, ―a” is the number of species in one habitat and ―b” is the number of species in the other habitat. The Jaccard‘s coefficient (SIJ) is a similarity measure and ranges between 0 and 1. When both habitats have same number of species, then SIJ = 1 whereas SIJ = 0 when the two habitats are far and distant. A Jaccard‘s similarity coefficient closer to 1 has a greater similarity to the ground truth, whereas a coefficient value closer to 0 has nearly no overlap area similar to the ground truth.

Bray-Curtis index of dissimilarity was used to calculate total population difference between two different sites, based on counts at each site.

BC = Σ |Xij - Xik| /Σ(Xij + Xik)

Where BC is the Bray–Curtis dissimilarity, ―Xij‖ is total individual in sample ―j‖, and ―Xik‖ is total individual in sample ―k‖. The Bray–Curtis dissimilarity is bound to be between 0 and 1, where ―1‖ means the two sites do not share any species and ―0‖ means the two sites share all the species. ―BC‖ is intermediate (e.g., BC = 0.5) at that sites where both habitats share some species (Bray and Curtis 1957). Calculating bacterial densities over the different observations was approached using relative abundance ―RA‖.

3.5: Isolation and identification of bacteria from alimentary tract of cockroaches

3.5.1: Collection of samples

Randomly selected 160 cockroaches (50 from hospitals and 110 from houses) observed for external bacterial infection were also subjected to isolation and identification of bacteria from alimentary tract.

3.5.2: Isolation of bacteria from alimentary tract of cockroaches

After external washings, cockroaches were placed in flasks rinsed with 70% ethanol for 5 min (to decontaminate external surfaces as 70% alcohol is bactericidal), transferred to sterilized flasks, and allowed to dry at room temperature under sterile conditions. Cockroaches were then washed with sterile normal saline for 2–3 min to remove traces of alcohol. Only cockroaches captured whole and live were utilized for this study. Each cockroach was dissected out and its mid gut was macerated aseptically in a sterile pestle and mortar in 2 ml of sterile normal saline.

3.5.3: Identification of microbes from alimentary tract of cockroaches

3.5.3.1: Preparation of media

Various types of media i.e., selective media, enrichment media and differential media were prepared to identify different bacterial isolates. Tryptic Soy Agar (TSA Lab M limited Cat # Lab11), Mannitol Salt Agar (MSA Himedia Cat # M118), MacConkey Agar (Merck, Germany Cat # VM 327265), Eosine Methylene Blue Agar (Lab M limited Cat # Lab 61), Salmonella Shigella Agar (SS agar Bio-Rad Cat # 64514 France), Blood Agar (Sigma Aldrich Cat #70133) were prepared for isolation and purification of bacterial colony.

3.5.3.2: Inoculation of sample

Aliquots (0.01 ml) of the macerate saline were then separately inoculated onto the surface of different selective and differential agar plates described in the above section. The agar plates were incubated at 37°C for 24 hrs.

3.5.3.3: Identification of microbes

The obtained colonies were identified as mentioned above by using standard bacteriological procedure as described by Burgey’s Manual of Determenative

Microbiology. The bacteria grown on the agar media were identified by microscopic, colonial morphology, Gram Staining (Cowan and Steel, 1974) and different biochemical tests e.g. oxidase test, catalase test, coagulase test and IMViC test (Hensyl, 1994).

3.5.4: Statistical analysis of microbial isolates from alimentary tract of cockroaches

Bacterial infection rate for each site was calculated for both species of cockroaches in relation to internal bacterial infections.

Bacterial infection rate = Total number of bacteria isolated X 100 Total infected cockroaches Calculations using Shannon-Wiener‘s diversity index (H′) (Shannon and Wiener, 1949) were conducted to measure the bacterial community diversity (all species of bacteria) in each habitat:

H′ = −ΣPilnPi

Pi = ni/N where, ―Pi‖ is the proportion of the specific bacterial species in the total sample, ―N‖ is the number of species richness.

Jaccard‘s index of similarity was used to analyze the similarity of the bacterial community among habitats (Jaccard, 1912). p = c/ (a + b + c)

Where, ―p” is Jaccard‘s index of similarity, ―c” is the number of the shared bacterial species, ―a” is the number of species in one habitat, ―b” is the number of species in another habitat. The Jaccard‘s coefficient (SIJ) is a similarity measure and ranges between ―0‖ and ‗1‖. When both habitats have same number of species, then SIJ = 1 whereas SIJ = 0 when the two habitats are far and distant. A Jaccard‘s similarity coefficient closer to 1 has a greater similarity to the ground truth, whereas a coefficient value closer to 0 has nearly no overlap area similar to the ground truth.

Bray-Curtis index of dissimilarity was used to calculate total population difference between two different sites, based on counts at each site.

BC = Σ |Xij - Xik| /Σ(Xij + Xik)

Where BC is the Bray–Curtis dissimilarity, ―Xij‖ is total individual in sample ―j‖, and ―Xik‖ is total individual in sample ―k‖. The Bray–Curtis dissimilarity is bound between 0 and 1, where1 means the two sites do not share any species and 0 means the two sites share all the species. ―BC‖ is intermediate (e.g., BC = 0.5) at that sites where both habitats share some species (Bray and Curtis 1957). Calculating bacterial densities over the different observations was approached using relative abundance ―RA‖.

3.6: Isolation of fungal flora from cockroaches

3.6.1: Preparation of samples

The set of 60 randomly selected cockroaches (30 P. americana and 30 B. germanica) collected from 10 houses were immobilized by keeping them in freezer at -4°C for 5 min. 2 ml of sterile formal saline was added to each test tube containing one cockroach, and the tubes were shaken thoroughly for 2 min to isolate fungal spores from the external surface of cockroach. Then cockroach was transferred to the other sterile tube with forceps (Evans and Richrdson, 1989). The washing was cultured on Sabouraud dextrose agar and Malt Extract agar supplemented with Terramycin (Oxytetracycline HCl) (250mg) for culturing fungal isolates.

3.6.2: Preparation of media for fungal culturing

General purpose media that are commonly used for fungal culture are Sabouraud dextrose and malt extract medium. To prevent contamination of the medium by bacteria, Terramycin was used during preparation, which permits the successful isolation of fungi and yeasts while inhibiting the growth of bacteria.

3.6.2.1: Preparation of Sabouraud’s Dextrose Agar (SDA)

Sabouraud Dextrose Agar (SDA) (Sigma Aldrich Cat # S3181) was a selective medium primarily used for the isolation of dermatophytes, other fungi and yeasts. The SDA media comprised of enzymatic digest of casein and animal tissues which provide a nutritious source of amino acids and nitrogenous compounds for the growth of fungi and yeasts. Dextrose was the fermentable carbohydrate incorporated in high concentration as a carbon and energy source while agar acts as solidifying agent.

65 gm of SDA was suspended in 300 ml of double distilled H2O water in a flask (Appendix VII). After completely dissolving the media final volume was made upto 1

60 litre by adding double distilled water. For proper dissolving the medium was heated with frequent agitation and boiled for one minute; autoclaved at 121° C for 15 min and after autoclaving Terramycin (Oxytatracycline HCl) (250 mg) capsule was added and mixed gently. After media were cooled down to 45-50°C then it was poured into petri dishes for further testing.

3.6.2.2: Preparation of Malt Extract Agar (MEA)

MEA (Himedia Cat # RM0048) is designed to contain the proper formulation of carbon, protein and nutrient sources used for the detection, isolation and enumeration of yeasts and molds. Dextrose is added to the medium to provide a carbon and energy source for fungi. Additionally, MEA contains digests of animal tissues (peptones) which provide a nutritious source of amino acids and nitrogenous compounds for the growth of molds and yeasts. Terramycin (Oxytetracycline HCl) (250 mg) was added to inhibit bacterial overgrowth while permitting successful selective isolation of fungi and yeasts.

50 gm of MEA powder was suspended in 300 ml distilled water and thoroughly mixed (Appendix VIII). For dissolving the powder completely, solution was heated with frequent agitation and boiled for 1 min. Final volume was made upto 1 litre by adding distilled water. The media was sterilized by autoclaving at 121°C for 10 min. To adjust the pH at 3.5, medium was cooled to 55°C and approximately 2-3 ml of 10% lactic acid/100 ml of MEA was added. Terramycin (Oxytetracycline HCl) (250 mg) was added immediately before pouring into the sterile Petri plates in order to suppress the bacterial growth.

3.6.3: Inoculation of samples

In order to culture fungal samples, 0.01 ml formal saline solution was inoculated with a sterile Pasture pipette in the center of SDA plates and MEA plates. Inoculum was spread over the media surface using spreader and allowed to diffuse into the agar for 10 min. The plates were incubated at 25-30°C in non-inverted position in an incubator with increased humidity for 10-14 days. Plates were examined frequently during each week for fungal growth and held for 30-35 days before being reported as negative. Discrete fungal colonies exhibiting typical color and morphology were examined to identify different fungal isolates.

3.6.4: Identification of fungal isolates

The fungal isolates were identified on the basis of color of colony, morphological or cultural characters (mycelial, vegetative and reproductive hyphae and spores) at First Fungal Culture Bank of Pakistan (FFCBP, Institute of Agricultural Sciences, University of the Punjab, Lahore Pakistan) up to species level by standard mycological methods (Zeini et al., 2002; Merz and Hay, 2007). Prevalence of each fungal isolate was calculated and prevalence of fungal contaminants for both species of cockroaches was calculated for all collection sites.

3.7: Isolation of parasitic contaminants from external surface of cockroaches

3.7.1. Preparation of samples

Isolation of parasitic ova/cyst from external surface of cockroaches was carried out by using formalin-ethyl acetate sedimentation method (Nyarango et al., 2008). Only those captured cockroaches were used in this study that were whole and live at the time of capturing. The set of 250 randomly selected cockroaches from 5 hospitals (65 P. americana and 60 B. germanica) and 5 houses (65 P. americana and 60 B. germanica) were immobilized by freezing at 0°C for 5 min. 2ml of sterile normal saline was added to each conical bottom centrifuge tube containing one cockroach, and the tubes were shaken vigorously for 2 min to isolate parasitic contaminants from the external surface of cockroach. Then cockroach was transferred to the other sterile tube with forceps. Isolation of parasitic cyst was carried out by centrifuging (90-2 centrifuge, Taizhou Juhao Import & Export Co., Ltd. China) 1 ml of normal saline washing at 2000 rpm for 5 min. Supernatant was discarded and sediments were stored for further investigations.

3.7.2. Identification of parasitic contaminants

The deposit was transferred to a clean glass slide and covered with a cover slip and the sediments were examined after staining with a drop of 1% Lugol iodine under 40X and 10X light microscope objectives. The parasites were identified and counted (Beaver et al., 1984; Al-Bayati et al., 2011). The deposit of fluid was stained by using

Ziehl Nelson stains for identifying characteristic features indicated by Cheesbrough (1993).

3.7.3. Statistical analysis

The parasite burden on the external surface of cockroaches in the two environmental settings for both species of cockroaches was calculated by using Shannon-Wiener‘s diversity index (H′) (Shannon and Weaver, 1949) and Simpson‘s index (Simpson, 1949).

Shannon-Wiener‘s diversity index was conducted to measure the parasitic contaminant diversity (for both species of cockroaches) in each habitat:

H′ = −ΣPilnPi

Pi = ni/N

Where, ―Pi‖ is the proportion of the specific parasitic protozoan/ helminthes species in the total sample and ―N‖ is the number of species richness.

Shannon-Wiener Diversity Index was calculated in order to determine the species diversity in different habitats as the presence of one individual of a species is not necessarily indicative of the species being present in a large number. Values of the Shannon's diversity index for real communities typically fall between 1.5 and 3.5.

Diversity index (D) = Σ(Pi)2

Simpson Diversity index = 1-D

Mean, standard deviation, probability was determined by one-way analysis of variance (ANOVA) by using SPSS (IBM SPS Statistics 20). Significance was set at P< 0.05 value.

3.8. Evaluation of antimicrobial sensitivity

The Kirby-Bauer‘s disk diffusion method was used to check the antibiotics susceptibility pattern of isolated bacteria. Commercially available antibiotics used in this study were Ceftriaxone (CRO) 30 µg (Oxoid CT0417B lot: 1316387),

Ciprofloxacin (CIP) 5 µg (Oxoid CT0425B. lot: 1312585), Amoxicillin (AML) 10 µg (Oxoid CT0161B lot: 1181136), Cephradine (CE) 30 µg (Oxoid CT0063B lot: 1213906) and Tetracycline (TE) 30 µg (Oxoid CT0054B lot: 1337826). Class of each antibiotics used in this study is enlisted in Table 3.8.

Table 3.8: List and class of antibiotics used in the present study.

S. No. Antibiotic Antibiotic Class

1 Ceftriaxone (CRO) 30 µg Cephalosporins (Cell wall synthesis inhibitor)

2 Ciprofloxacin (CIP) 5 µg Fluoroquinolone (DNA synthesis inhibitor)

3 Amoxicillin (AML) 10 µg Penicillins (Cell wall synthesis inhibitor)

4 Cephradine (CE) 30 µg Cephalosporins ( Cell wall synthesis inhibitor)

5 Tetracycline (TE) 30 µg Protein synthesis inhibitor

3.8.1. Preparation of Tryptic Soy Broth (TSB)

Tryptic Soy Broth (TSB Lab M limited Cat # Lab4) was a nutritious medium that supported the growth of a wide variety of microorganisms, especially common aerobic and facultatively anaerobic bacteria.

30 gm TSB powder was weighed and dispersed in 300 ml of double distilled water (Appendix XI). After dissolving completely, final volume was made upto 100 ml with double distilled water. 10 ml of media was dispensed into each screw cap glass test tubes and autoclaved at 121ºC for 15 min then cooled to room temperature.

3.8.2: Inoculation of sample in Tryptic Soy Broth (TSB)

In sterile environment within the range of laminar flow cabinet (Heraguard™ ECO series, Thermo Scientific), pure colony of bacterial isolates were picked from culture plates of Mannitol Salt agar (MSA), MacConkey agar, Eosine methylene blue agar (EMB), Salmonella Shigella agar (SSA) and Blood agar plate (BAP) with the help of inoculating loop. The picked bacterial colony was suspended in autoclaved TSB test tubes and shake well. The test tubes were incubated at 37°C for 24 hrs to get enough bacterial growth for further analysis.

3.8.3: Preparation of Mueller Hinton Agar plates

Mueller Hinton agar is a non-selective, non-differential medium. Mueller and Hinton developed Mueller Hinton Agar (MH agar) in 1941 for the isolation of pathogenic Neisseria species. Nowadays, it is more commonly used for the routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique and its performance is specified by the CLSI. Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch and Agar.

38 gm of the MH agar powder (Merck Germany Cat # VL978437) was suspended in 300 ml of distilled water. Heated with frequent agitation and boiled for one minute to completely dissolve the medium in distilled water. Final volume was made upto 1 litre by adding distilled water and autoclaved at 121°C for 15 min then cooled to room temperature. Cooled MH Agar was poured into sterile petri dishes on a smooth, horizontal surface to give uniform depth and media was allowed to cool to room temperature. Plates were stored at 2-8 ºC for further use.

3.8.4: Inoculation of samples on Muller Hinton Agar

Sterile swabs were used to pick the inocula from 24 hrs bacterial culture in TSB (Lab

M limited Cat # Lab004) and streaking was done over the entire sterile surface of MH agar plate. The streaking was repeated 2-3 times by rotating the plate at 60º each time to ensure uniform distribution of inoculums. The antimicrobial disks of specific concentration were dispensed onto the medium surface gently using sterile forceps.

Each disk was pressed down enough to come in contact with agar surface and was incubated for 24 hrs at 37°C.

3.8.5: Result analysis

Diameters of zones of inhibition around the discs were measured to the nearest millimeter (mm) using a meter ruler, and the isolates were classified as sensitive (S) and resistant (R) according to the standardized table supplied by the CLSI guidelines

(2013) (Appendix XXIII). A clear zone indicates that bacteria are sensitive to that antibiotic while absence of clear zone indicates resistance of bacteria against the particular antibiotic.

3.9: Efficacy of common disinfectants for bacterial isolates

In order to determine efficacy of different disinfectants against microbial isolates 3 disinfectants were tested. These were: Germ kill vantocil IB {Poly (Hexamethylene

65 biguanide) hydrochlorides (PHMB) 11% (ARCH biocide Blackley, Manchester, UK. Batch: 786101)}, G-cide as crystal HLD {Glutarol 9.8% vantocil GA (ARCH biocide Blackley, Manchester, UK. Batch: 09XY345064)} and Germ Kill Vantocil FHC {Poly (Hexamethylene biguanide) hydrochloride (PHMB) 20% (ARCH biocide Blackley, Manchester, UK. Batch: 786010)}. Well diffusion method was used for 3 different dilutions of each disinfectant to assess its inhibition zone diameter and minimum inhibitory concentration (MIC). MIC of each disinfectant was calculated as the lowest concentration that did not give any visible bacterial growth usually expressed in µg/mL or mg/L (Appendix XXIV).

3.9.1: Preparation of MH Agar plates

MH agar plates were prepared in a similar way as previously described in the above described text.

3.9.2: Inoculation of samples

Sterile swabs were used to pick the inoculums from 24 hrs bacterial culture in TSB and streaking was done over the entire sterile surface of MH agar plate with sterile swabs. The streaking was repeated 2-3 times by rotating the plate at 60º each time to ensure uniform distribution of the inoculum over the entire agar surface of MH agar plate.

3.9.3: Serial dilution of disinfectants

The efficacy of disinfectants was determined by dilution method using serially diluted disinfectants according to the NCCLS protocol (NCCLS, 2000). The Germ kill vantocil IB, G-cide as crystal HLD and Germ Kill Vantocil FHC were diluted to get series of concentrations from 12.5%, 25% and 50% (v/v) in sterile nutrient broth. The lowest concentration of disinfectant that inhibited the growth of the bacterium on MH agar plate was considered as the MIC.

3.9.4: Well diffusion method

Six holes per plate each with a diameter of 6-8 mm were punched aseptically with a sterile cork borer. Holes were labeled for each concentrationof all disinfectants. A volume of 20-100 μl of different dilutions of disinfectants (Germ kill vantocil IB, G- cide as crystal HLD and Germ Kill Vantocil FHC) was introduced into its respective holes and allowed to diffuse into medium. Negative control was performed by

66 introducing sterile broth in its well. The plates were then incubated at 37°C for 24 hrs. The disinfectants diffused into the medium to its extent and inhibit the growth of the inoculated microbial isolates. The resulting zones of inhibition were uniformly circular as there was a confluent lawn of growth. The diameter of zone of inhibition was measured in millimeters and interpreted as per the CLSI guidelines (2014). Antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the well (NCCLS, 1993). Relative inhibition zone diameter was calculated by applying the formula:

%RIZD = (IZD sample –IZD negative control) x 100

IZD disinfectants standard

Where, ―RIZD‖ denotes (relative inhibition zone diameter in mm) and ―IZD‖ denotes (inhibition zone diameter in mm)

It compensates the possible effect of the solvent (blank) other than water on the ―IZD‖. The resulting ―IZD‖ of the samples were either higher than or equal to the ―IZD‖ of the blanks. Therefore, the obtained percentages were positive. The test was considered negative (-) when the ―IZD‖ of the sample was equal to the ―IZD‖ of the blank.

Antibacterial activity of the disinfectant was observed as the growth of microbes on MH agar plate. Observations was calculated as ++ (heavy Growth of microorganisms), + (moderate growth of microorganisms) and - (no growth observed) for each concentration of tested disinfectant.

3.10: Quantitative and qualitative analysis of total protein extracted from resistant bacterial isolates

Resistant bacterial isolates in disk diffusion method and well diffusion methods were sequestered for molecular analysis. A total of 12 samples were selected for total protein extraction. Total protein was extracted from overnight grown bacterial culture at 37°C in brain heart infusion (Difco Laboratories) using Bacterial Protein Extraction Kit BS596 (Bio Basic Inc. Canada) (Appendix XXV).

3.10.1: Preparation of reagents

3.10.1.1: 1 X Phosphate buffer saline (PBS)

To prepare 1X PBS, 50 ml 10X PBS was added in 450 ml double distilled H2O. Mixed well and stored at room temperature (Appendix XXVI).

3.10.1.2: 1X cell lysis buffer

To prepare 1X cell lysis buffer, 200 µl 10X cell lysis buffer was added in 1800 µl ddH2O. Eppendorf was rotated between palms to mix well and stored at 4ºC (Appendix XXVII).

3.10.1.3: Phenylmethylsulfonyl fluoride (PMSF) solution

To prepare 80 µl PMSF solutions 0.8 µl PMSF solution was added into 79.2 µl isopropanol and mixed well and kept in eppendorf at •20ºC for future use (Appendix XXVIII).

3.10.2: Protein extraction procedure

10 ml grown culture was shifted into conical centrifuge tubes and centrifuged at 5,000 rpm (Eppendorf 5804R refrigerated benchtop centrifuge) for 10 min at 4ºC. Supernatant was removed and pellet was washed with 10 ml 1X PBS solution. Cell pellet was suspended in 40 µl of 1X cell lysis buffer [Tris (hydroxymethyl) aminomethane, ethylenediamine tetra-acetic acid and sodium dodecyl sulphate]. After dissolving cell pellet in cell lysis buffer, 4 μl of the PMSF (prevent protein lysis) solution and 8 μl of the Lysozyme solution was added in cell suspension and incubated on ice for 30 min. Mixture was incubated on a rocking platform (Brunswick innova 4230 refrigerated incubator shaker) for 10 min at 4°C. 2 μl of DNaseI/RNase solution was added to the mixture and incubation was continued on a rocking platform for another 10 min at 4°C. Mixture was centrifuged at 3,000 rpm (Eppendorf 5804R refrigerated benchtop centrifuge) for 30 min at 4°C and insoluble debris was removed. Supernatant (cell lysate) was collected in an eppendorf and stored at -20ºC until it was electrophoresed on SDS-PAGE (BIO RAD Mini PROTEAN® tetra cell).

3.10.3: Bradford protein assay

The Bradford protein assay was used to measure the concentration of total protein in a sample. Its principle based on the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue.

3.10.3.1: Preparation of Bradford reagent

50 gm of Coomassie Brilliant Blue G-250 was added in 50 ml of methanol and 100 ml

85% (w/v) phosphoric acid (H3PO4) was added to the mixture. Acid solution mixture was added slowly into 850 ml of H2O and left to dissolve completely (Appendix XXIX). To remove the precipitates the solution was filtered and stored at 4ºC (He, 2011).

3.10.3.2: Preparation of dilutions of standard protein sample {Bovine Serum Albumin (BSA)}

6 dilutions of BSA with a range of 50-2000 µg/ml were prepared with distilled water in test tubes and labeled properly. 30 µl unknown protein sample and dilutions of standard were aliquot in separate dry, clean test tubes and labeled appropriately. 30 µl of water was used as reagent blank in spectrophotometer (Unico SpectroQuest Model SQ2800 Single Beam).

3.10.3.3: Macro assay

To perform Macro assay, 5 ml Bradford reagent was added into each test tube, mixed well gently to avoid foaming. The mixture was incubated at room temperature for 5 min and absorbance was measured at 280 nm against reagent blank. Standard curve was drawn on Microsoft excel to determine unknown concentration of samples.

Figure 3.10.3.3: Standard curve for Bradford’ assay (Bradford, 1976).

3.10.3.4: Calculations for unknown samples

Concentration of unknown sample was calculated by using the following formula

y = 0.0321x-0.033

Where ―y‖ = unknown sample absorbance and ―x‖ = unknown sample concentration

3.10.4: SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was used for total protein separation and visualizing on gel. It consisted of resolving and Stacking Gel. 10% resolving gel and 5% stacking gel was prepared and poured in SDS-PAGE caster. After solidifying caster was placed in gel apparatus, 1X running buffer was added; protein samples were loaded and then run at 70 V for 1 hr.

3.10.4.1: Reagent preparation

3.10.4.1.1: 30% Acrylamide/Bisacrylamide solution

For preparing 30% acrylamide-bisacrylamide solution, 29.2 gm acrylamide and 0.8 gm Bis acrylamide were completely dissolved in small quantity of distilled water with the help of magnetic stirrer. When the clear solution was attained the total volume was made upto 100 ml with distilled water using volumetric cylinder. The solution was stored at 4ºC in a refrigerator (Appendix XXX).

3.10.4.1.2: Tris-HCl 1.5 M (pH 8.8) Resolving gel buffer

In order to prepare 1.5 M solution of Tris-HCl, 18.6 gm of Trizma base [Tris- Hydroxymethyl amino methane] was dissolved in small amount of distilled water using magnetic stirrer. The pH was adjusted to 8.8 using concentrated HCl. The final volume was made upto 100 ml with distilled water using volumetric cylinder. Tris- HCl was stored at 4ºC in refrigerator (Appendix XXXI).

3.10.4.1.3: 1M Tris-HCl (pH 6.8) Stacking gel buffer

Trizma base [Tris-hydroxymethyl amino methane] 12.11 gm was dissolved in little amount of distilled water with the help of a magnetic stirrer to make 1M Tris-HCl. The pH of the solution was adjusted to 6.8 using concentrated HCl. Final volume was made upto 100 ml with distilled water. Tris-HCl was stored in a refrigerator at 4ºC (Appendix XXXII).

3.10.4.1.4: 10% Sodium Dodecyl Sulphate (SDS) Solution

10 % SDS was prepared by dissolving 10 g of SDS in 80 ml of distilled water using a magnetic stirrer. Stirring of SDS produces massive bubbles. To settle the bubbles the solution was kept at room temperature for 3-4 hrs. Once the SDS was entirely

70 dissolved, the final volume was made upto 100 ml by adding distilled water and the ultimate solution was stored at room temperature (Appendix XXXIII).

3.10.4.1.5: 10% Ammonium per sulphate (APS) solution

Ammonium per sulphate (APS) 0.1 gm was dissolved in 1 ml of distilled water in an eppendorf tube. Each time 10% APS was prepared freshly (Appendix XXXIV).

3.10.4.1.6: 1X Running buffer (SDS Electrophoresis buffer)

1X of the running buffer was prepared by adding 18.8 gm Glycine, 3.02 Tris Base, 10ml of 10% SDS in 500 ml distilled water. Final volume of the solution was made upto 1 litre with distilled water by volumetric cylinder and stored at 4ºC (Appendix XXXV).

3.10.4.1.7: Tracking dye (loading dye)

154 mg of Dithiothreitol (DTT) and 200 mg of SDS were dissolved in 8 ml of 1M Tris (pH 6.8) and 10 ml of glycerol was dissolved in the mixture followed by the addition of 20 mg of Bromophenol blue dye and thoroughly mixed up. The prepared loading dye divided in 1.5 ml aliquots in eppendorf tubes which were covered with aluminium foils were stored at 4ºC (Appendix XXXVI).

3.10.4.1.8: Coomassie stain (Staining solution)

125 mg of Coomassie Blue R250 was taken in a stopper flask and 112.5 ml methanol, 22.5 ml acetic acid and 112.5 ml distilled water were added to the flask. The solution was dissolved completely using magnetic stirrer. Final staining solution was stored in a dark bottle at room temperature (Appendix XXXVII).

3.10.4.1.9: Coomassie destain (Destaining solution)

50 ml of methanol and 70 ml of acetic acid were mixed in a beaker. The final volume was made 1litre in a graduated cylinder using distilled water and the final solution was stored at room temperature (Appendix XXXVIII).

3.10.4.1.10: Preparation of working dilutions

Protein samples extracted by using bacterial protein extraction kit (Cat # BS596) were diluted with loading dye to prepare the working dilutions for SDS gel electrophoresis. For this purpose, 10 µl of loading dye was added in 10 µl of protein sample in labeled fresh eppendorf tubes. The contents of the tubes were mixed well by vortexing and

71 tubes were heated for 5 min in boiling water bath for the denaturation of sample proteins. 15 µl of each sample was loaded onto the polyacrylamide gel.

3.10.4.1.11: Protein Marker

10 µl of the Bench Mark Protein ladder by Life technologies [Cat # 10747012] were loaded in the well of gel. Ladder with different molecular weight reference bands revealed 14 molecular weight band ranges (Appendix XXXIX).

3.10.4.2: Gel preparation

For casting the gel for polymerization, the glass plates of gel casting apparatus (BIO RAD Mini PROTEAN® tetra cell) were assembled using 1 mm thick spacer. Bacterial proteins were finally resolved on 10% gel that was optimized after running different gel of 12%, 10% and 8% gels, respectively.

3.10.4.2.1: 10% Resolving gel preparation

10 ml of the 10% resolving gel was prepared by following the recipe mentioned in Appendix XL. The resolving gel solution was mixed thoroughly after adding TEMED and poured in between the glass plates assembled in the gel assembly leaving 1 cm area empty at the top for stacking gel. Almost 100 µl of iso-propanol was layered at the top of the gel to avoid dryness and to give a smooth surface and to remove oxygen from the surface of the gel as it inhibits polymerization. Gel was then left undisturbed at room temperature for about 20 min for polymerization.

3.10.4.2.2: 5% Stacking gel preparation

In the meantime resolving gel takes for polymerization, 2 ml of the 5% stacking gel was prepared by using recipe mentioned in Appendix XLI. After the polymerization of the resolving gel the overlying iso-propanol was removed by using the blotting paper. The stacking gel was poured over polymerized resolving gel. A comb was inserted in the stacking gel to prepare wells for sample loading. The gel was again left undisturbed at room temperature till polymerization.

3.10.4.3: Gel electrophoresis

The gel plates were disassembled from gel casting apparatus and fixed in the electrophoresis chamber (BIO RAD Mini PROTEAN® tetra cell) and placed in the electrophoresis tank filled with already prepared electrophoresis buffer to a volume so as the bottom of the gel was dipped in it. The central area of the electrophoresis

72 chamber was filled completely with the same buffer. Comb was gently removed in upward direction to keep the wells in proper shape. With the help of micropipette, 10 µl protein ladder was loaded in the first well and 15 µl of each sample was loaded in the consecutive wells. Electrophoresis was performed for about 1-1.5 hrs or until the time (15 min after) the dye seems to diffused in the buffer of lower chamber at a current supply of 170 V in a cooling chamber maintained at 4ºC.

3.10.4.4: Staining

After electrophoresis, the gel was removed from the plates very carefully. The two glass plates were separated from each other with the help of a spatula. The gel bed was transferred to the staining solution. The box containing the gel and stain was placed on a shaker for 30 min for continuous uniform staining of the gel.

3.10.4.5: Destaining

The gel was washed with distilled water and then immersed into the destaining solution and kept on shaker for constant shaking until background became transparent and protein bands became visible as blue colored light and dark bands.

3.10.4.6: Image capture and photography

After destaining, the gel was photographed and images were saved for further analysis by TotalLab Quant V11.5.

3.10.4.7: Quantification for protein fractions

The quantification of electrophoretically separated protein fractions was carried out by TotalLab Quant V11.5 that provided the data based on the molecular density of each fraction. The data were analyzed and employed in finding changes in proteins levels in resistant bacterial isolates in comparison with the susceptible bacterial isolates.

RESULTS

4.1: Collection and Identification of Cockroaches

4.1.1: Identified species of cockroaches

From different types of human dwellings 4 species of cockroaches belonging to two families (Blattidea and Blattelidae) were identified during the entire sampling period.

1. American cockroach (Periplaneta americana):

This cockroach species is reddish brown in appearence with a yellowish digit 8 pattern on the light -olored pronotum and commonly reaches a length of nearly 51mm (Fig. 4.1.1a). The young nymphs of this species are greyish brown turning reddish brown after a few molts. They occur in sewers and basements especially around pipes and drains (Fig. 4.1.1e).

2. Oriental cockroach (Blatta orientalis):

The adult male of this species is shiny attaining body length of 25mm with wings covering three-quarter of the body length, leaving the last few abdominal segments exposed. The females of this species are wingless and are also shiny black but larger than males with body length of 32mm. Both male and female are incapable of flight. The nymphs of B. orientalis are uniformly reddish brown to black. They species inhabit decaying filthy materials outdoor beneath the mulch, leaf litter, stones and debris outdoors, and garbage (Fig. 4.1.1c).

3. German cockroach (Blattela germanica):

This cockroach species has light brown body color and two dark stripes on the pronotum with an average body length of 12.5mm. Both males and females of this species have wings extending to the end of their abdomen but they do not fly (Fig. 4.1.1b). They occurs in food storage areas especially kitchens (Fig. 4.1.1f).

4. Turkestan cockroach (Blatta (Shelfordella) lateralis (Walker):

Turkestan cockroaches are native to a large area of the Middle East extending from Libya eastward to central Asia including Afghanistan, Pakistan, Uzbekistan and southern Russia (Alesho, 1997). The male and females of this species are strikingly different. The small brownish yellow male size is 12cm and its pronotum has a pale 74

margin as do the upper corner of the front wings. The female is similar to oriental cockroach female in appearance with shiny dark black body with short wings (Fig. 4.1.1d). However, the outer margin line of wing pads is pale like the males of this species. They are found in compost piles, gardens, potted plants and homes with clay floors (Fig. 4.1.1g).

Fig. 4.1.1a-4.1.1d: Pictorial presentation of different cockroach species collected from Lahore, Pakistan.

Fig. 4.1.1a: American cockroach {P. americana (A: female, B: male)} Fig. 4.1.1b: German cockroach {B. germanica (A: male, B: female laying ootheca, C: nymph)} Fig. 4.1.1 c: Oriental cockroach {B. orientalis (A: female laying ootheca, B: male)} Fig. 4.1.1 d: Turkestan cockroach {B. lateralis (A: male, B: female)} 75

Fig. 4.1.1e-g. Cockroach infested sites in different houses of Lahore, Pakistan.

Fig. 4.1.1e: Kitchen cabinet infested with B. germanica

Fig. 4.1.1f: Emergence of nymph from ootheca showing heavy infestation in a house

Fig. 4.1.1g: Basement of house infested with P. americana showing ootheca and newly molted nymph. 76

4.1.2: Distribution and abundance of cockroach species

This study revealed that B. germanica was the most dominant species belonging to family Blattelidae comprising of 45% of the total collection followed by P. americana (35%) belonging to family Blattidae. These two species comprises 80% of the total specimens collected. The other two species included B. orientalis and B. (Shelfordella) lateralis (Walker) respecrtively forme 9% and 11% of the total specimens trapped. From all the specimens captured during the study period 8328 specimen (62%) were collected at different nymphal stages, 3626 (27%) were adult males and 1479 (11%) were adult females of total catch. The number of females was lower as compared to males in traps because females are less agile and are often hidden in deep crevices, engaged in reproductive activity.

4.1.2.1: First Trimester: During the first trimester (April-June, 2013) of the study average minimum and maximum air temperatures were 23.8°C and 37.6°C, respectively, and relative humidity was 41% (Table 4.1.2a). The spring was reaching its end and summer was starting. This was the most favorable season for breeding of eggs and nymphs. In this trimester, nymph collection was more prominent as compared to adult males and females. B.germanica was the most dominant species (46.78%) of all catches, followed by P. americana (33.50%) B. orientalis (9.96%) and B. lateralis (9.75%), respectively (Table 4.1.2b). Diversity indices were worked out for all species found in 20 different sites revealed Shannons index value of 1.178691 and Simpson’s index of diversity was 0.6495. B. germanica with a value of 0.218879 (Simpson’s index) and 0.35542 (Shannon index) was the dominant species during the first trimester of the study.

4.1.2.2: Second Trimeser: During the second trimester of study (July-September, 2013) the average daily minimum and maximum air temperatures were 24.8°C and 34.1°C and relative humidity was 74% (Table 4.1.2a). Summer season was at peak and cockroaches were mostly found in indoors sheltered areas. This was the most favorable time for metamorphosis of nymphs to adults. Different cockroach species had different life spans and time spans to turn from nymphs to adults. In this trimester, the last nymphal stage predominated in collections compared to adult males and females. B. germanica was the most dominant species (44.36%) in all catches, followed by P. americana (35.81%). B. orientalis and B. lateralis comprised 8.11% 77

and 11.7%, respectively (Table 4.1.2b). Diversity indices for this trimester revelaed Shannons index value of 1.183401 and Simpson’s index of diversity was 0.6547 for this trimester. B. germanica was the dominant species in this period with a value of 0.196748 (Simpson’s index) and 0.36062 (Shannon index). During this trimester higher number of nymphs of late instars were recorded, they had gone through metamorphosis to change into adults and were ready for reproduction. In most species time for late instars to turn into females is longer thanthe time required for corresponding males.

4.1.2.3: Third Trimester: In third trimester (October-December, 2013) of the study B. germanica was the most predominant species (44.62%), followed by P. americana, B. orientalis and B. lateralis respectively. Table 4.1.2b shows the Shannon index value of (1.178746) and Simpson’s index of diversity (0.6521) for all species of cockroaches collected from different sites. Both species richness and abundance were significantly lower during colder months of the study period.

4.1.2.4: Fourth Trimester: In the fourth trimester of study (January-March, 2014) Shannon index value was 1.207116 and Simpson’s index of diversity was 0.6687. Blattela germanica was most dominant species (40.82%), followed by P. americana (37.02%). Blatta orientalis (7.17%) and B. lateralis (14.97%) (Table 4.1.2b). Overall species evenness and richness was found higher in the fourth trimester of study.

Table 4.1.2a: Average minimum and maximum air temperatures and relative humidity (%) recorded from April 2013 to March 2014 (four trimesters). Trimester Average Temperature Average No. of Percentage (°C) relative cockroaches % humidity trapped Minimum Maximum (%)

1st (April- June) 23.8 37.6 41 4696 35 2nd (July – Sep) 24.8 34.1 74 5927 44 3rd (Oct– Dec) 13 26.5 68 1862 14 4th (Jan – March) 9 21.4 68 948 7

Table 4.1.2b: Diversity indices of different cockroach species collected during four trimesters of sampling (April 2013– March 2014).

Trimester Species No. of % age Relative Shannon Simpson index Species evenness cockroaches abundance index (S) H/logS ΣPi2 Pi Pi(lnPi) 1st P. americana 1573 33.50 0.3349 -0.3664 0.112202 1.1787 /0.60206 = 1.957764 B. germanica 2197 46.78 0.4678 -0.35542 0.218879 B. orientalis 468 9.96 0.0996 -0.22984 0.009932 B. lateralis 458 9.75 0.0975 -0.22703 0.009512 Total 4696 H= 1.1787 D= 0.3505, 1-D= 0.6495 2nd P. americana 2123 35.81 0.358191 -0.36779 0.128301 1.1834 /0.60206 = 1.965586 B. germanica 2629 44.36 0.443563 -0.36062 0.196748 B. orientalis 481 8.11 0.081154 -0.20383 0.006586 B. lateralis 694 11.7 0.117091 -0.25116 0.01371 Total 5927 H= 1.1834 D= 0.3453, 1-D= 0.6547 3rd P. americana 671 36.03 0.360365 -0.36784 0.129863 1.178746/0.60206 = 2.470538 B. germanica 831 44.62 0.446294 -0.3601 0.199179 B. orientalis 162 8.70 0.087003 -0.21247 0.00757 B. lateralis 198 10.63 0.106337 -0.23834 0.011308 Total 1862 H= D= 0.3479, 1.178746 1-D= 0.6521 4th P. americana 351 37.02 0.370253 -0.36791 0.137087 1.207116/0.60206 = 2.529999 B. germanica 387 40.82 0.408228 -0.36578 0.16665 B. orientalis 68 7.17 0.07173 -0.18902 0.005145 B. lateralis 142 14.97 0.149789 -0.28441 0.022437 Total 948 H= D= 0.3313, 1.207116 1-D= 0.6687

Some previous studies have described P. americana and B. orientalis as outdoors species of cockroaches but can intrude indoors environments through sewerage pipes and crevices during harsh seasons. Also, some previous studies had reported that B. germanica and B. lateralis were always found in indoors environments (Jeffery et al. 2012, Rust and Reierson, 2007). In the present study residential areas/houses and hospitals were mostly infested with B. germanica. Periplaneta americana enter outdoors environments through sewerage pipes and holes. Blatta orientalis and B. lateralis are morphologically similar but their colonies can be identified by their male members. Houses and hospitals were highly infested with P. americana and B. germanica as compared to offices, shopping mall/departmental stores and universities. Whereas, B. orientalis was commonly found in houses, institutes/universities, followed by hospitals, offices and shopping malls/ departmental stores. Distribution of B. lateralis was most common in institutes/ universities, houses and offices with basements and gardens (Table 4.1.2c).

4.1.2.5: Relative abundance

Relative abundance of total collected cockroaches belonging to different species differed significantly among different seasons and appeared to be related to air temperature. Mean number (1179.50 + 351.77) of P. americana was found to be higher in all trimesters with analysis of variance revealing a significant difference between the groups (F (3,4) = 9.65, P=0.005); the mean and Standard deviations values are presented in Table 4.1.2d. Population peak of cockroaches was observed in second trimester (summer season) and the least occurred in fourth trimester (winter season) (Table 4.1.2a). Although the abundance of cockroache’s differed among different human sampling localities, the fluctuated seasonal fluctuation trend was similar in all different sampling localities. As a generalization, it could be stated that the relative abundance of cockroaches are determined by the habitat conditions especially average temperatures and humidity determining the seasonal fluctuations in relative abundance of cockroaches in that area. Lower population levels were observed during cold weather conditions while high levels of populations of cockroaches were observed during moderate to hot seasons.

Table 4.1.2c: Sampling location types and number of cockroaches belonging to different species trapped from the various experimental sites. Location type Number of Number of Infested Number (%) of identified locations sampling units sampling units cockroaches Hospitals 5 86 77 (89.5%) P. americana (36%) B. germanica (55%) B. orientalis (7%) B. lateralis (2%) Dormitories in 4 75 60 (80%) P. americana (39%) Universities B. germanica (41%) B. orientalis (10%) B. lateralis (10%) Houses 7 130 122 (93.8%) P. americana (38%) B. germanica (39%) B. orientalis (10%) B. lateralis (13%) Offices 1 22 19 (86.3%) P. americana (25%) B. germanica (39%) B. orientalis (10%) B. lateralis (26%) Departmental 3 30 17 (56.6%) P. americana (23%) stores B. germanica (54%) B. orientalis (5%) B. lateralis (18%)

Table 4.1.2d: Air temperature, humidity, mean, standard deviation and one-way ANOVA values of different cockroach species collected in different trimesters from urban locations in Lahore, Pakistan.

Cockroach Mean + S.E SD value SS df Mean square F Significance species at 5% level (P) Air 23.75 + 7.544 temperature 2.619

Relative 62.50 + 14.663 3311724.95 3 1103908.32 9.65 0.005 humidity 6.17

P. Americana 1179.50 + 814.457 351.77 2460622.30 12 205051.86 B. germanica 1511.00 + 1071.805 470.61 B. orientalis 294.75 + 211.142 88.09 B. lateralis 353.25 + 259.563 112.24

Some environmental factors such as average air temperature and relative humidity showed significant correlation with population changes and abundance of P. americana in the study sites (Pearson correlation, r= 0.904, P= 0.04) (Table 4.1.2e). Similarly air temperature had positive correlation with populations of B. germanica (r= 0.958, P= 0.021) and B. orientalis (r= 0.987, P= 0.007). Another factor that can be pointed as an effective factor on infestation rate of cockroaches in different habitats is age of the infrastructure. However, non-significant correlation was observed between average air temperature and B. lateralis (Table 4.1.2e). Pearson correlation between relative humidity and different species of cockroaches was negative but statistically non-significant for P. americana, B. germanica and B. orientalis. The correlation of cockroach species with climatic conditions indicated that biodiversity and population dynamics of cockroaches was significantly correlated with maximum temperature, minimum relative humidity, and to considerable extent with age of building (infrastructure) and maintenance of hygiene standard.

Post hoc comparisons using the Fisher LSD test revealed that populations of P. americana and B. germanica had significantly greater difference with average temperature than B. orientalis and B. lateralis; however B. orientalis and B. lateralis did not differ significantly from the average temperature (Table 4.1.2f). 82

Table 4.1.2e: Coefficient of correlation between environmental factor (air temperature and relative humidity) and populations of different cockroach species collected from urban areas of Lahore, Pakistan.

P. americana B. germanica B. orientalis B. lateralis Temperature r = .904* r = 0.958* r = .987* r = .834 ns, P = 0.04 p = 0.021 P = 0.007 P = 0.083 Humidity r = -.115 ns, r = -.227 ns, r = -.359 ns, r = .066 ns, P= 0.442 P = 0.387 P = 0.321 P = 0.467 * = Correlation is significantly different at 0.05 level (1- tailed) of significance, ns = non-significant

Table 4.1.2f: Post hoc comparisons using the Fisher LSD test for the one-way analysis of variance between air temperature and populations of different cockroach species collected in Lahore, Pakistan.

Sr. no. Comparison* difference of Mean T 1 1:2 -1155.75 6.252 2 1:3 -1482.25 8.045 3 1:4 -271.00 1.466 4 1:5 -329.50 1.782 *1 = average temperature, 2 = P. americana, 3 = B. germanica, 4= B. orientalis, 5 = B. lateralis **Least significant difference (Scheffe) for p(0.05)=691.7039 ***Least significant difference (Tukey's Honestly Significant Difference): for p<0.05 =853.5780

4.2: Isolation of microorganisms from external and internal surfaces of cockroaches

In this study, 160 cockroaches were observed for the presence of bacterial flora. Randomly selected 50 cockroaches from hospitals and 110 cockroaches from different houses were observed for internal and external bacterial infection. This was done to investigate the bacterial infection among two groups of cockroaches.

4.2.1: Identified bacterial species

The dominant bacterial species in the 16 experimental sites of hospitals and houses were E. coli, S. aureus (Fig. 4.2.1i), S. typhi, S. dysentriae (Fig. 4.2.1g.), P. aeruginosa, S. epidermidis (Fig. 4.2.1h), P. mirabilis, P. vulgaris, E. aerogenes, S. pneumoniae, B. cereus, K. pneumonia (Fig. 4.2.1c), E. faecalis and E. cloacae. They were identified using colony morphology; gram staining and biochemical tests for complete identification (Table 4.2.1a) (Fig. 4.2.1j- 4.2.1k). The most common diagnostic bacterium isolated from external surface of cockroaches was E. coli (10.31%) (Fig. 4.2.1a- 4.2.1b). The second common diagnosed bacterium isolated from external surface of cockroaches was S. aureus (Fig. 4.2.1e) and third common diagnosed bacterium was P. mirabilis with relative frequency of 10.09% and 8.78%, respectively. In contrast, the most common diagnostic bacterium isolated from internal gut tract of cockroaches was found to be P. aeruginosa (Fig. 4.2.1f) with relative frequency of 19.96%, while P. vulagaris and E. faecalis were found as second and third most abundant bacterium isolated from gut tract of cockroaches with relative frequency of 16.08% and 14.60% respectively (Table 4.2.1b). No S. typhi, S. epidermidis, E. aerogenes and S. pneumonia (Fig. 4.2.1d) were found in digestive tract of any cockroach.

It was observed that P. americana were more infected than B. germanica. All cockroaches were found to be infected with at least one bacterium. Microbes isolated from external and internal surfaces of cockroaches indicated high prevalence of bacteria on external surface rather than in the digestive tract. However, the most abundant externally and internally isolated microbes were also different indicating that both surfaces have different pathological impact for their carrier. This could be 84

explained by the enteric gram-negative bacteria being better adapted to invade the gut, having stable and nutritious conditions superior to those on the exterior of the insect.

Fig. 4.2.1.a-b: Petri plates showing pure lawn growth of E. coli. Fig. 4.2.1a: Growth of E. coli on EMB agar plates. Fig. 4.2.1b: Growth of E. coli on MacConkey agar plates.

Fig. 4.2.1c: K. pneumoniae pure culture growth on MacConkey (left) and EMB agar (right) plates. 85

Fig. 4.2.1d-e: Mannitol Salt agar plates showing growth of bacteria. Fig. 4.2.1d: Pure growth lawn of S. pneumonia. Fig. 4.2.1e: Pure growth lawn of S. aureus.

Fig. 4.2.1f: EMB agar plate showing pure growth of P. aeruginosa. Fig. 4.2.1g: Salmonella Shigella agar plate showing growth of S. dysentriae. 86

Fig. 4.2.1h: Mannitol salt agar plate showing growth of S. pyogenes and S. epidermidis.

Fig. 4.2.1i: Nutrient agar plate showing growth of S. aureus.

Figure 4.2.1j: Biochemical test results of Citrate test. Figure 4.2.1k: Biochemical test results of Catalase test. 87

Table 4.2.1a: Details of identification and biochemical characters used for classical identification of bacterial isolates from cockroaches collected in Lahore, Pakistan. Bacterial Microscopic colonial Gram Oxidase Coagulase Catalase IMViC test species morphology staining test test test Indole MR VP Citarate test test E. coli Circular, entire, raised, -ve rod _ N/A _ + + _ _ punctiform small, translusent S. aureus Circular, entire, convex, +ve cocci _ + + _ + + + moderate large, opaque. S. typhi Entire, convex, shiny -ve rod _ N/A + _ + _ + colonies S. Circular, convex, , smooth, -ve rod _ N/A _ _ + _ _ dysentriae ranslucent P. Umbonate, medium size, -ve rod + N/A + _ _ _ + aeruginosa mucoid coloniy flagellate S. Circular, entire, convex, +ve cocci _ _ + + _ + _ epidermidis pinhead colonies, whitish P. mirabilis Medium size, lobate, -ve rod _ N/A + _ + _ + raised, opaque P. vulgaris Medium, punctiform, -ve rod _ N/A + + + _ _ convex, undulating E. Entire, convex, shiny -ve rod _ N/A + _ _ + + aerogens colony S. Circular, entire, raised with +ve ______pneumonia depressed in center coccus e K. Complete, round, raised, -ve rod _ N/A + - + - + pneumonia translucent, mucoid e B. cereus Irregular, , opaque with +ve rod + _ + _ _ + + rough matted surface flagellate d E. faecalis Convex, pinpoint colonies +ve cocci _ _ _ _ _ + _ E. cloacae Smooth, round, rough -ve rod _ N/A + _ _ + + "cauliflower" type colonies flagellate d

(-) indicates negative test, (+) indicates positive test, (N/A) indicates test not applicable for this species.

Table 4.2.1b: Relative abundance of bacterial isolates harbored on external and internal body surfaces of cockroaches (n=110) collected in Lahore, Pakistan. Bacterial isolate External surfaces Internal surfaces Abundance Frequency Abundance Frequency E. coli 142 10.31% 31 5.73% S. aureus 139 10.09% 43 7.95% S. typhi 108 7.84% - - S. dysentriae 93 6.75% 13 2.40% P. aeruginosa 113 8.20% 108 19.96% S. epidermidis 103 7.48% - - P. mirabilis 121 8.78% 30 5.54% P. vulgaris 79 5.73% 87 16.08% E. aerogens 35 2.54% - - S. pneumoniae 104 7.55% - - K. pneumoniae 120 8.72% 68 12.57% B. cereus 114 8.28% 44 8.13% E. faecalis 73 5.30% 79 14.60% E. cloacae 33 2.40% 38 7.02% Total 1377 100 541 100

4.2.2: Bacterial infection rate in hospitals and houses

Randomly picked 110 cockroaches collected from houses and 50 from various hospitals were observed for external and internal bacterial loads. Among hospitals, it was found that in P. americana highest external bacterial infection was observed in PIC (75.6%) while highest internal bacterial infection was observed in SZH (30.8%). Same trend was observed for B. germanica in which highest external bacterial contamination was found in PIC (32.4%) and highest intestinal bacterial contamination was observed in SZH (66%). Overall, P. americana collected from SZH had highest bacterial infection (98.7%) while lowest was found in JH (87.3%). Highest bacterial infection rate for B. germanica was observed for PIC (96.9%) and lowest for JH (68.7%) (Table 4.2.2a).

Among houses highest external bacterial infection (55.9%) for P. americana was observed in samples collected from SHL-II while highest external infection (52%) for B. germanica was also from SHL-II locality. Highest internal contamination (25.8%) 89

for P. americana was observed in MUG-I locality while highest internal infection (28.8%) for B. germanica was MOD-III house. Collectively, P. americana from MUG-I (76.5%) had highest bacterial infection and MOD-II (52.4%) had lowest infection while B. germanica from MOD-III (80.3%) had highest bacterial contamination and MOD-I (48.2%) had lowest bacterial contamination (Table 4.2.2b).

Table 4.2.2a: External and internal bacterial infection rate of hospital cockroaches in different hospitals of Lahore, Pakistan. Hospital American cockroach (n= 35) Total German cockroach (n= 15) Total name* (%) Internal External Internal External (%) infection (%) infection (%) infection (%) infection (%) SH 23.1% 72.1% 95.2% 49.5% 30.9% 80.4% PIC 18.9% 75.6% 94.5% 64.5% 32.4% 96.9% SZH 30.8% 67.9% 98.7% 66% 29.1% 95.1% GH 21% 66.5% 87.5% 45% 28.5% 73.5% JH 18.2% 69.3% 87.3% 39% 29.7% 68.7% *(SH=Services Hospital, PIC=Punjab Institute of Cardiology, SZH=Sheikh Zayed Hospital, GH=General Hospital, JH=Jinnah hospital)

Table 4.2.2b: External and internal bacterial infection rate of cockroaches collected from different houses of Lahore, Pakistan. Houses* American cockroach (n= 57) Total German cockroach (n= 53) Total (%) Internal External Internal External (%) infection (%) infection infection (%) infection (%) (%) SHD-I 23.8% 47.8% 71.6% 20.2% 44.5% 64.7% SHD-II 21.4% 50.2% 71.6% 20.6% 46.6% 67.2% MUG-I 25.8% 50.7% 76.5% 13.2% 47.2% 60.4% MUG-II 17.1% 47.3% 64.4% 15.9% 43.2% 59.1% JT-I 19.7% 48.5% 68.2% 18.3% 45.1% 63.4% JT-II 16.4 50.1% 66.5% 15.6% 49.6% 65.2% SHL-I 19.7% 45.6% 65.1% 18.3% 42.4% 60.7% SHL-II 15.6% 55.9% 71.5% 14.4% 52% 66.4% MOD-I 16.1% 36.5% 52.6% 14.9% 33.3% 48.2% MOD-II 12.5% 39.9% 52.4% 11.5% 37.1% 48.6% MOD-III 19.2 55.8% 75% 28.8 51.5% 80.3% *(SHD=Shadman colony, MUG=Mughalpura, JT=Johar town, SHL=Shalamar town, MOD=Model town)

4.2.3: Ecological indices of isolated microorganisms

Jaccard’s index of similarity of bacterial species found on cockroaches was highest (0.3125) in houses (SHD-II, JT-I and MOD-I) indicating that most bacterial species were isolated from cockroaches collected from houses. Whereas, Jaccard’s index of similarity of bacterial species found on cockroaches was highest for houses (0.3125) and lowest (0.2813) for hospitals with PIC having the least bacterial species on cockroaches in houses. Similarly, Bray-Curtis index of dissimilarity was highest for PIC (0.2174) indicating more bacterial species there and lowest Bray-Curtis index of dissimilarity for three houses (0.0909) SHD-II, MOD-I and JT-I, indicating similar species composition for all three habitats. The highest Shannon-Wiener’s diversity index value was found in bacteria on cockroaches collected from GH (2.274247), while the lowest Shannon-Wiener’s diversity index (1.968198) was found in PIC (Table 4.2.3).

Table 4.2.3: Community diversity indices of bacterial species in and on cockroaches collected from different habitats (hospitals and houses) in Lahore, Pakistan. Habitat Habitat No. of bacterial species Jaccard’s Bray-Curtis Shannon- type name* Index of index of Wiener’s diversity External surface Internal surface similarity dissimilarity index Hospitals SH 14 10 0.294117647 0.166666667 2.055202 Hospitals PIC 14 9 0.2813 0.2174 1.968198 Hospitals SZH 14 10 0.294117647 0.166666667 2.070408 Hospitals GH 14 10 0.294117647 0.166666667 2.274247 Hospitals JH 14 10 0.294117647 0.166666667 2.011843 Houses SHD-I 12 9 0.3 0.142857 2.078622 Houses SHD-II 12 10 0.3125 0.090909 2.146585 Houses MUG-I 12 9 0.3 0.142857 2.0759 Houses MUG-II 12 9 0.3 0.142857 2.087459 Houses JT-I 12 10 0.3125 0.090909 2.141777 Houses JT-II 12 9 0.3 0.142857 2.111517 Houses SHL-I 12 9 0.3 0.142857 1.981644 Houses SHL-II 12 9 0.3 0.142857 2.101056 Houses MOD-I 12 10 0.3125 0.090909 2.167071 Houses MOD-II 12 9 0.3 0.142857 2.004845 Houses MOD-III 12 9 0.3 0.142857 2.067839 *(SH=Services Hospital, PIC=Punjab Institute of Cardiology, SZH=Sheikh Zayed Hospital, GH=General Hospital, JH=Jinnah hospital, SHD=Shadman colony, MUG=Mughalpura, JT=Johar town, SHL=Shalamar town, MOD=Model town). 91

4.3: Isolation of fungal flora from cockroaches 4.3.1: Variety of fungal isolates

Fungal isolation from 60 samples of cockroaches (P. americana and B. germanica) was done for 10 houses. The cockroaches were caught from kitchens, bathrooms and bedrooms in these houses. All samples had one or more species of medically important mold on external surface. The most common molds isolated and identified by FFCBP were Geotrichum candidum (G. candidum) (22%) (Fig. 4.3.1a), Aspergillus flavus (A. flavus) (75%) (Fig. 4.3.1b), Metarhizium anisopoliae (M. anisolpoliae) (20%) (Fig. 4.3.1e), Metarhizium spp. (14%), Aspergillus oryzae (A. oryzae) (84%) (Fig. 4.3.1h) and Aspergillus zonatus (A. zonatus) (50%) (Fig. 4.3.1d). Other medically important molds, Alternaria alternate (A. alternata) (Fig. 4.3.1c), A. niger (Fig. 4.3.1g), Mucor spp. (Fig. 4.3.1f), Trichoderma spp. and Penicillium spp. were rarely isolated from few cockroaches.

Identification of fungal isolates was done by both color and morphological features of colony and other microscopic characteristics such as hyphae, spores, conidia and conidiophore of fungi (Table 4.3.1) (Fig 4.3.1i-p). FFCBP allotted accession numbers to fungal isolates mentioned in Table 4.3.1. Microscopic characters helped in identifying fungal strains upto species level.

Table.4.3.1: Prevalence of fungal contaminants isolated from cockroaches (n=60) and their identification characteristics. Fungal FFCBP n (%) Color of colony Morphological characteristics isolates accession no# Geotrichum 1306 13 (22%) Flat, white to cream, dry and Hyphae are hyaline, septate, branched. candidum finely suede-like with no Arthroconidia are 6-12 x 3-6 µm in size reverse pigment. and are released by the separation of a double septum. Aspergillus 1307 45 (75%) granular, flat, often with radial Conidial heads are typically radiate, flavus grooves, yellow at first but later splitting to form loose columns quickly becoming bright to (mostly 300-400 µm in diameter), dark yellow-green with age biseriate. Conidia are globose to subglobose (3-6 µm in diameter), pale green. Metarhizium 1308 12 (20%) Mostly light green, white edge Conidia varied in size, cylindrical to anisopoliae of variable thickness oval, runcae at both ends Metarhizium 1309 8 (14%) Mostly velvety, dark green, Conidia varied in size, cylindrical to spp. light green, white or brownish, oval, often slightly narrowed in the white edge of variable middle, usually runcae at both ends thickness Aspergillus 1310 50 (84%) Wet, powdery, white center Conidial heads radiate to loosely oryzae and green yellow in periphery columnar, vesicles pyriform to subglobose, uniseriate often biseriate, conidia smooth to finely roughened, globose to ellipsoidal. Aspergillus 1311 30 (50%) Conspicuous concentric zones, Conidial heads radiate to loosely zonatus bright yellow for 3-4 days, columnar, conidiophore are thin walled, ecru-olive when mature and colorless and smooth, vesicles typically reverse to colorless or slightly globose brown.

Fig. 4.3.1a-d: Culture plates of fungal isolates grown on Malt Extract agar (MEA). Fig. 4.3.1a: Geotrichum candidum colony grown on Malt Extract agar. Fig. 4.3.1b: A. flavus colony grown on Malt Extract agar plate. Fig. 4.3.1c: A. alternata colony grown on Malt Extract agar plate. Fig. 4.3.1d: A. zonatus colony grown on Malt Extract agar plate.

Fig. 4.3.1e-h: Culture plates of fungal isolates grown on Malt Extract agar (MEA). Fig. 4.3.1e: M. anisopoliae colony grown on Malt Extract agar plate. Fig. 4.3.1f: Mucor spp. grown on Malt Extract agar plate. Fig. 4.3.1g: Aspergillus spp. grown on Malt Extract agar plate. Fig. 4.3.1h: A. oryzae colony grown on Malt Extract agar plate.

Fig. 4.3.1i-k: Micrographs of conidia of fungi isolated from external surfaces of cockroaches. Fig. 4.3.1i: Micrograph of conidia of A. flavus isolated from external surfaces of cockroaches. Fig. 4.3.1j: Micrograph of conidia of A. oryzae isolated from external surfaces of cockroaches. Fig. 4.3.1k: Micrograph of conidia of G. candidum isolated from external surfaces of cockroaches. 96

Fig. 4.3.1l-n: Micrographs of conidiophore of fungi isolated from cockroaches.

Fig. 4.3.1l: Micrograph of conidiophore of A. flavus isolated from external surfaces of cockroaches.

Fig. 4.3.1m: Micrograph of conidiophore of A. oryzae isolated from external surfaces of cockroaches.

Fig. 4.3.1n: Micrograph of conidiophore of Metarhizium spp. isolated from external surfaces of cockroaches. 97

Fig. 4.3.1o-p: Micrographs of conidiophore of fungi isolated from external surfaces of cockroaches.

Fig. 4.3.1o: Micrograph of conidiophore of G. candidum isolated from external surfaces of cockroaches.

Fig. 4.3.1p: Micrograph of conidiophore of A. zonatus isolated from external surfaces of cockroaches. 98

4.3.2: Prevalence of fungal flora isolated from P. americana and B. germanica

Two species of collected cockroaches P. americana and B. germanica from houses were observed for fungal contamination. Prevalence of fungal contaminants was found different in both species; 85% of total P. americana and 53% of total B. germanica were contaminated by one or more fungal spores. Contamination prevalence for each site was also different as revealed by Table 4.3.2. P. americana and B. germanica collected from SHD1, JT1 and SHL2 were highly contaminated with fungal spores and showed relatively high prevalence, while P. americana and B. germanica showed lowest prevalence of fungal contaminants in SHD2 and MUG2. Issac et al. (2014) isolated seven fungi from Benin, Africa 6 fungi from Ekpoma and 6 from Emuhi; A. niger were highest in Benin and Ekpoma, while Mucor spp. was found highest in Emuhi. Rhizopus spp. were not found in Ekpoma and Emuhi and were found in Benin only. This difference could have been due to the number of cockroaches analyzed for each site.

Table.4.3.2: Prevalence of fungal contaminants in cockroaches collected from various houses of Lahore, Pakistan. Collection P. americana (30) B. germanica (30) site* No. of Number +ve for %age No. of Number +ve for %age cockroaches fungal cockroaches fungal examined contaminants examined contaminants SHD-I 3 3 100% 3 2 66% SHD-II 3 1 33% 3 2 66% MUG-I 3 3 100% 3 1 33% MUG-II 3 2 66% 3 1 33% JT-I 3 3 100% 3 2 66% JT-II 3 2 66% 3 2 66% SHL-I 3 3 100% 3 1 33% SHL-II 3 3 100% 3 2 66% MOD-I 3 2 66% 3 2 66% MOD-II 3 3 100% 3 1 33% *(SHD=Shadman colony, MUG=Mughalpura, JT=Johar town, SHL=Shalamar town, MOD=Model town)

4.4: Isolation of parasitic contaminants from cockroaches

4.4.1: Identified species of parasites isolated from cockroaches

Results of this study showed that 112 (44.8%) cockroaches harbored at least one human intestinal parasite on their body surfaces. The cockroaches from hospital environment harbored more parasites than houses. 47 (33.57%) cockroaches from houses and 65 (59.09%) from hospitals were infected with parasitic organisms. In the isolated parasitic contaminants, 76 (67.85%) cockroaches harbored parasitic protozoans and 36(32.15%) carry pathogenic and non-pathogenic intestinal parasites. Viable eggs and dormant cysts of parasites can hitch a ride on cockroaches. Ova and cysts of parasitic organism may settle into the crevices and cracks between thorax and head. There are so many fissures and clefts and crannies on a cockroach which provide site for these organisms.

P. americana harboured more parasites as compared to B. germanica in both environment. Most common human intestinal parasites found on cockroaches include ova of A. lumbricoides (giant roundworm) (Fig. 4.4.1a), T. trichura (whipworm) (Fig. 4.4.1e), A. duodenalae (hookworm), E. vermicularis (pinworm) (Fig. 4.4.1b-c), Taenia spp. and S. stercoralis (threadworm (Fig. 4.4.1d)). The cysts of protozoans’ parasites including B. coli, E. histolytica (Fig. 4.4.1f), C. parvum (Fig. 4.4.1g), I. belli from cockroaches. Both cockroach species carry almost same parasitic species on their external surfaces. E. coli protozoan was found as the most prevalent in both sites followed by E. vermicularis and E. histolytica. A. lumbricoides were least prevalent in hospitals (52%) and houses (43%) for P. americana and B. germanica.

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Fig. 4.4.1a-d: Pictorial representation of parasites isolated from cockroaches. Fig. 4.4.1a: A. lumbricoides eggs isolated from body surface of cockroaches. Fig. 4.4.1b: E. vermicularis eggs isolated from body surface of cockroaches. Fig. 4.4.1c: E. vermicularis larvae isolated from body surface of cockroaches. Fig. 4.4.1d: Larvae of S. stercoralis isolated from body surface of cockroaches. 101

Fig. 4.4.1e-h. Pictorial representation of parasites isolated from cockroaches. Fig. 4.4.1e: Ova of T. trichura isolated from body surface of cockroaches. Fig. 4.4.1f: Cysts of E. histolytica isolated from body surface of cockroaches. Fig. 4.4.1g: Oocyst of C. parvum isolated from body surface of cockroaches. Fig. 4.4.1h: Oocyst of I. belli isolated from body surface of cockroaches.

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4.4.2: Ecological indices of parasitic contaminants of cockroaches

As Simpson Diversity Index can limit diversity value for minor sample size, therefore it is principally suitable for rapidly evaluating regions for conservation (Lande et al., 2000). Simpson Diversity index value of parasitic contaminants isolated from B. germanica collected from houses was highest with value of 0.92133 and that indicated that B. germanica had the lowest diversity of parasitic contaminants (Table 4.4.2a) (Fig. 4.4.2a). While Simpson Diversity index for B. germanica collected from hospital was 0.91827 which indicated higher diversity than P. americana. The former cockroach species (B. germanica) found in indoor sites of hospitals and had more chances to encounter with filthy habitats and faecal waste in bathrooms (Table 4.4.2b) (Fig. 4.4.2b).

The Shannon-Weiner Diversity Index specifying the comparative occurrence of many species and used to associate species abundance and relative richness amongst species was found to be highest for P. americana at both sites, houses as well as hospitals with values of 2.554291 and 2.536765 respectively which indicates that the rate of parasitic contaminants of both species was not even (Table 4.4.2a-b). These values lying in the range of the Shannon-Weiner Diversity Index predicting the community has diversity in the parasitic contamination carried by cockroaches. These variations in parasitic contamination can be explained as both cockroach species have varied ecology, habitat and chance to encounter with parasites while foraging for food.

Both experimental sites were not significantly different in carriage of parasitic contaminants on cockroaches (F (1,6) =1.795, P= 0.229). An analysis of variance revealed that no significant difference was observed among the parasitic components of two species of cockroaches (F (1,6) =5.032, P= 0.066) and also no significant difference was observed among the helminthes parasites and protozoan parasitic component (F (1,6) =0.459, P= 0.523) while mean and standard deviation values are presented in Table 4.4.2c.

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Table 4.4.2a: Prevalence and ecological indices of parasitic contaminants isolated from P. americana and B. germanica collected from different houses of Lahore, Pakistan. Parasite species Houses n(A,B)=65,60 Relative abundance Shannon index Simpson index

No. (%age) Pi Pi(lnPi) ΣPi2

A* B** A B A B A B

A. lumbricoides (ova) 15 (23) 12 (20) 0.060976 0.058038 -0.17058 -0.16523 0.003718 0.003368

T. trichura (ova) 19 (29.2) 17 (28.3) 0.077236 0.082124 -0.19781 -0.20529 0.005965 0.006744

A. duodenale (ova) 15 (23) 19 (31.6) 0.060976 0.091701 -0.17058 -0.21912 0.003718 0.008409 E. vermicularis (ova/ 20 (30.7) 19 (31.6) 0.081301 0.091701 -0.20405 -0.21912 0.00661 0.008409 larvae)

Taenia spp. (ova) 20 (30.7) 16 (26.6) 0.081301 0.077191 -0.20405 -0.19774 0.00661 0.005958

S. stercoralis (ova) 18 (27.7) 14 (23.3) 0.073171 0.067615 -0.19136 -0.18217 0.005354 0.004572

B. coli (cyst) 18 (27.7) 15 (25) 0.073171 0.072548 -0.19136 -0.19035 0.005354 0.005263

E. histolytica (cyst) 20 (30.7) 18 (30) 0.081301 0.087057 -0.20405 -0.21255 0.00661 0.007579

C. parvum (oocyst) 17 (26.1) 15 (25) 0.069106 0.072548 -0.18468 -0.19035 0.004776 0.005263

I.belli (oocyst) 20 (30.7) 15 (25) 0.081301 0.072548 -0.20405 -0.19035 0.00661 0.005263

G. duodenalis (cyst) 19 (29.2) 13 (21.6) 0.077236 0.062681 -0.19781 -0.17363 0.005965 0.003929

C. cayetenensis (cyst) 18 (27.7) 14 (23.3) 0.073171 0.067615 -0.19136 -0.18217 0.005354 0.004572

E. coli (cyst) 27 (41.5) 20 (33.3) 0.109756 0.096634 -0.24253 -0.22584 0.012046 0.009338

Total Species Evenness A= 0.436107 D= 0.07869, D= 0.07867, 1- Species evenness B = 2.292663 H= 2.554291 H= 2.553896 1-D= 0.92131 D= 0.92133

*A= P. americana, **B= B. germanica

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Table 4.4.2b: Prevalence and ecological indices of parasitic contaminants isolated from P. americana and B. germanica collected from different hospitals in Lahore, Pakistan. Parasite species Hospitals Relative abundance Shannon index Simpson index n(A,B)=65,60 Pi Pi(lnPi) ΣPi2 No. (%age)

A B A B A B A B

A, lumbricoides (ova) 23 (35.4) 10 (16.6) 0.057257 0.046827 -0.16378 -0.14337 0.003278 0.002193

T. trichura (ova) 39 (60) 18 (30) 0.097047 0.084626 -0.22639 -0.20901 0.009418 0.007162

A. duodenale (ova) 27 (41.5) 19 (31.6) 0.067124 0.08914 -0.18133 -0.21552 0.004506 0.007946 E. vermicularis (ova/ 38 25 (41.6) larvae) (58.46) 0.094556 0.117348 -0.22304 -0.25146 0.008941 0.013771

Taenia spp. (ova) 26 (40) 21 (35) 0.064698 0.098731 -0.17716 -0.22862 0.004186 0.009748

S. stercoralis (ova) 25 (38.5) 14 (23.3) 0.062272 0.065726 -0.1729 -0.17894 0.003878 0.00432

B. coli (cyst) 27 (41.5) 12 (20) 0.067124 0.056417 -0.18133 -0.16222 0.004506 0.003183

E. histolytica (cyst) 40 (61.5) 21 (35) 0.099473 0.098731 -0.22959 -0.22862 0.009895 0.009748

C. parvum (oocyst) 30 (46.2) 16 (26.6) 0.074726 0.075035 -0.19385 -0.19435 0.005584 0.00563

I. belli (oocyst) 27 (41.5) 13 (21.6) 0.067124 0.060931 -0.18133 -0.1705 0.004506 0.003713

G. duodenalis (cyst) 24 (36.9) 13 (21.6) 0.059684 0.060931 -0.16825 -0.1705 0.003562 0.003713

C. cayetenensis (cyst) 27 (41.5) 15 (25) 0.067124 0.070522 -0.18133 -0.18703 0.004506 0.004973

E. coli (cyst) 49 (75.3) 16 (26.6) 0.121793 0.075035 -0.25645 -0.19435 0.014834 0.00563

Total Species Evenness A= 2.277284 D= 0.08160, D= 0.08173, Species evenness B = 2.275234 H= 2.536765 H= 2.534482 1-D= 0.91840 1-D= 0.91827

*A= P. americana, **B= B. germanica

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Fig. 4.4.2a: Prevalence of parasites isolated from the P. americana and B. germanica collected from different houses in Lahore, Pakistan.

Fig. 4.4.2b: Prevalence of parasites isolated from the P. americana and B. germanica collected from different hospitals in Lahore, Pakistan.

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Table 4.4.2c: Mean, Standard deviation, one way ANOVA of parasitic isolates from cockroaches collected from different houses and hospitals in Lahore, Pakistan. Factors Mean + S.E. Standard SS df MS F Significance deviation at 5% level (SD) (P)

Houses 113.25 + 9.02 18.043 3280.500 1 3280.500 1.795 0.229

Hospitals 153.75 + 8.85 57.702 6 1827.583

P. americana 162.00 +25.25 50.510 1 6498.00 5.032 0.066

B. germanica 105.00 + 2.79 5.597 6498.00 6 1291.33

Helminthes 122.25 + 8.73 37.464 1 1012.500 0.459 0.523

Protozoans 144.75 + 7.42 58.841 1012.500 6 2205.583

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4.5: Evaluation of antimicrobial sensitivity

Drug sensitivity was tested for 100 isolates of 7 species of pathogenic bacteria. Multidrug resistant pattern was observed in almost all isolates. Resistance to amoxicillin was found to be 100% for both gram-negative isolates; E. coli (Figure 4.5b), S. typhi (Figure 4.5c), P. aeruginosa ( Figure 4.5.d) and P. mirabilis) and gram positive isolates (S. aureus) ( Figure 4.5.a). Highest resistance for both gram negative and gram-positive bacterial isolates to amoxicillin was followed by resistance to cephradine and tetracycline, respectively (Table 4.5.a & b). Ciprofloxacin is one of the most powerful quinolones against gram-negative bacteria, including methicillin- resistant and anaerobic staphylococci. Overall resistance to ciprofloxacin (fluoroquinolones) was observed much lower for all isolates. The most frequent pattern of resistance observed in this study was AML > CE > TE > CRO > CIP.

Ceftriaxon (CRO) is a third-generation semi-synthetic cephalosporin that acts upon gram-positive bacilli and gram-negative bacilli and is not affected by beta-lactamase. It can act on gram-negative bacilli that are resistant to first- and second-generation cefalosporins, however, there may be resistance as a result of the non-hydrolytic barrier, impermeability mechanisms, and modification of their action receptor or by penicillin-fixing proteins. Cephradine is a first-generation cefalosporin antibiotic that is characterized by its bactericidal activity on gram-negative and gram-positive bacteria.

Amoxycillin is a moderate-spectrum, bacteriolytic, β-lactam antibiotic in the aminopenicillin family used to treat susceptible Gram-positive and Gram-negative bacteria. It can be absorb easily after oral administration than other β-lactam antibiotics. Tetracycline a broad-spectrum antibiotic exhibiting activity against a wide range of gram positive and gram negative bacteria and protozoan parasites. They are protein synthesis inhibitors, inhibiting the binding of aminoacyl-tRNA to the mRNA- ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex.

E. coli and P. aeruginosa are most resistant to commonly used antibiotics. They achieve antibiotic resistance through a combination of antibiotic uptake through outer membrane and a variety of energy dependent mechanisms (Table 3.8). E. coli was observed as resistant to 3 out of 5 antibiotics (AML, CE, TE), followed by P. 108

aeruginosa that showed resistance to amoxicillin and tetracycline. S. aureus, S. typhi, P. mirabilis and K. pneumonia exhibited resistance to amoxicillin and cephradine, respectively. S. dysentriae revealed sensitivity towards different drugs which can be explained by the ecological niche of this isolate.

Fig. 4.5a-d: Mueller Hinton agar plates showing antimicrobial sensitivity patterns. Fig. 4.5a: Antimicrobial sensitivity pattern of S. aureus. Fig. 4.5b: Antimicrobial sensitivity pattern of E. coli. Fig. 4.5c: Antimicrobial sensitivity pattern of S. typhi. Fig. 4.5d: Antimicrobial sensitivity pattern of P. aeruginosa.

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Table 4.5a: Antibiotic sensitivity profile of bacterial isolates obtained from cockroaches (P. americana and B. germanica). Bacterial No. of CRO CIP AML CE TE

isolate isolates No. (%) No. (%) No. (%) No. (%) No. (%)

E. coli 25 17 (68) 22 (88) - - -

S. aureus 15 11 (74) 3 (20) - 7 (46.7) 10 (66.6)

S. typhi 10 8 (80) 8 (80) - 6 (60) 6 (60)

S. dysentriae 10 7 (70) 8 (80) 5 (50) 6 (60) 8 (80)

P. aeruginosa 20 13 (65) 17 (85) - 9 (45) -

K. pneumonia 10 5 (50) 7 (70) 3 (30) - 6 (60)

P. mirabilis 10 6 (60) 8 (80) - 4 (40) 4 (40)

Ceftriaxone: CRO, Ciprofloxacin: CIP, Amoxicillin: AML, Cephradine: CE, Tetracycline: TE

Table 4.5b: Antibiotic resistance profile of bacterial isolates obtained from cockroaches (P. mericana and B. germanica). Bacterial No. of CRO CIP AML CE TE

isolate isolates No. (%) No. (%) No. (%) No. (%) No. (%)

E. coli 25 8 (32) 3 (12) 10 (100) 10 (100) 10 (100)

S. aureus 15 4 (26) 12 (80) 10 (100) 8 (53.3) 5 (33.4)

S. typhi 10 2 (20) 2 (20) 10 (100) 4 (40) 4 (40)

S. dysentriae 10 3 (30) 2 (20) 5 (50) 4 (40) 2 (20)

P. aeruginosa 20 7 (35) 3 (15) 10 (100) 11 (55) 20 (100)

K. pneumonia 10 5 (50) 3 (30) 7 (70) 10 (100) 4 (40)

P. mirabilis 10 4 (40) 2 (20) 10 (100) 6 (60) 6 (60)

Ceftriaxone: CRO, Ciprofloxacin: CIP, Amoxicillin: AML, Cephradine: CE, Tetracycline: TE

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4.6: Efficacy of common disinfectants for bacterial isolates

Susceptibility of 7 drug-resistant bacterial isolates was investigated against 3 commercial disinfectants commonly applied in hospitals and houses. Inhibitory effect was observed as high, mild or low by measuring the inhibition zone diameter. Mean of inhibition zone diameter was calculated for each concentration of disinfectant against all drug resistant bacterial isolates. Bactericide effect depends on diffusion process of disinfectants in the media. The diffusion process depends on numerous factors including number, size and shape of particles. An important factor is number of particles as the disinfectant diffuse faster at higher gradient of concentration. Another factor is particle volume which influences the diffusion rate, such as small particles will diffuse faster and large ones will diffuse slower. As the molecule radius increases one expects diffusivity to decrease proportionally to radius-squared because of solute-solvent increased interactions. Another neglected factor is temperature also, as temperature rises diffusion process increased because of increased average kinetic energy of molecules (Heneine, 2000). Besides polarity of samples, the pH of solvents can also affect the results of bactericides, since microbial growth might be hindered in media which have been rendered too acid or too alkaline by samples.

Germ kill Vantocil FHC exhibited highest inhibition zone diameter mean (27+11.575) for 12.5% diluton and 14+13.856 at 50% dilution. Similarly, RIZD was 81.81%, 54.54% and 4.54% for 12.5%, 25% and 50% dilutions, respectively. G-cide as crystal HLD had the indicating highest inhibition zone diameter (26.66+4.61) at 50% dilution which was relatively lower than Germ Kill Vantocil FHC indicating its lower efficacy compared to RIZD 70%, 40% and 10% at 12.5%, 25% and 50% dilutions respectively (Table 4.6a). Germ Kill Vantocil IB showed highest mean of inhibition zone diameter (22.11+1.68) at 50% dilution and recorded as the lowest efficacy with RIZD -9.18. This negative value indicated that its inhibition zone diameter was less than IZD of control group. The outcome of this study proves Germ kill Vantocil FHC to be the strongest antimicrobial agent irrespective of the dilutions when compared with the other disinfectants used in this study. Disparity from recommended dose of disinfectant may be due to the water from different sources used to dilute the disinfectants often containing dissolved impurities and other organic substances that might interfere with the efficacy of the disinfectants. However, the storage of diluted 111

disinfectant solution in large containers for longer periods to use later may lead to the observed levels of bacterial contamination.

Table 4.6a: Means + SE values of inhibition zone diameter using 3 different concentrations of 3 different disinfectants against bacteria isolated from cockroaches. Bacterial Zones of growth inhibition diameter (mm) isolate Germ kill vantocil IB G-cide as crystal HLD Germ Kill Vantocil FHC

12.5% 25% 50% 12.5% 25% 50% 12.5% 25% 50% (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v)

E. coli 16.14+0. 14.28+0 17.6+3 24.6+4.0 21.2+5.6 16.14+6. 84 .94 21+1.02 19+3.64 .31 2 1 20+4.87 59 S. aureus 14.77+2. 12.11+1 22.11+1 19.42+2 17.14+ 25.11+3. 15.6+4. 14.77+3. 38 .90 .68 .63 3.76 36 20+3.90 44 71 S. typhi 13.6+0. 12.4+0 22.8+2. 20.4+2.3 16+1.41 14+1.67 12+1.67 98 .75 10+1.10 25.2+2.8 15 2 S. dysentriae 12.66+1 26.66+4. 24+11.0 14+1.76 12+2 16+3.06 .15 8+0.67 61 27+2.40 5 14+13.86 P. aeruginosa 21.66+2 21.33+ 20.5+8.9 17.5+7.2 15+1 13+1 11+1 .73 2.40 5 27+3 21+8.60 2 K. pneumoniae 18.25+1. 17.25+0 15.5+0. 22.66+6 21.33+ 19.33+5. 27.5+11. 22.5+9. 03 .75 96 .68 6.25 60 23 30 24+12 P. mirabilis 17.33+2 13.33+3 20+8.6 27+11.5 22+3.06 .40 .33 24+1.53 4 17+7.26 8 22+9.93 14+8.08

% RIZD 45.45 18.18 -9.18 70 40 10 81.81 54.54 4.54

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Efficacy analysis of antibacterial activity of surface disinfectants revealed that antibacterial activity of various disinfectants varied for each isolate. MIC of Germ kill Vantocil FHC for E. coli (Fig. 4.6b) and K. pneumonia obtained at 50% dilution, while S. aureus (Fig. 4.6a), S. typhi, S. dysentriae, P. aeruginosa and P. mirabilis showed resistance at this high concentration of Germ Kill Vantocil FHC. However, on the contrary G-cide as crystal HLD exhibited MIC for S. typhi and S. dysentriae at its highest concentration (50%). Bactericidal activity was not observed for other isolates but bacteriostatic activity was noticeable for E. coli, S. aureus, P. aeruginosa and K. pneumonia. Germ kil Vantocil IB demonstrated MIC for only one isolate (S. typhi) but showed remarkable bacteriostatic activity for S. dysentriae, P. aeruginosa and K. pneumoniae at all concentrations. Overall, S. aureus, P. aeruginosa and P. mirabilis exhibited highest resistance towards test disinfectants while S. dysentriae showed least resistance to all isolates (Table 4.6b). Similar findings were observed by Okore et al. (2014) where Dettol had broad spectrum activity as it inhibited the growth of gram-positive (S. aureus and Streptococcus spp.) and gram-negative (E. coli) bacteria. The antimicrobial activity was more on the gram-positives isolates (Streptococcus spp. (30mm) and S. aureus (28mm)).

Fig. 4.6a: Zones of inhibition of disinfectants used against S. aureus in well diffusion method. Fig. 4.6b: Zones of inhibition of disinfectants used against E. coli in well diffusion method.

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Table 4.6b: Effectiveness of three disinfectant agents on bacteria isolated from cockroaches. Bacterial Antibacterial activity of surface disinfectants (++, +, -) isolate Germ kill vantocil IB G-cide as crystal HLD Germ Kill Vantocil FHC

12.5% 25% 50% 12.5% 25% 50% 12.5% 25% 50% (v/v) (v/v) (v/v)a (v/v) (v/v) (v/v)b (v/v) (v/v) (v/v)c

E. coli ++ + + ++ + + ++ + _

S. aureus ++ + + ++ + + ++ ++ +

S. typhi ++ + _ + + _ ++ ++ +

S. dysentriae + + + + + _ ++ + +

P. aeruginosa + + + ++ + + ++ ++ +

K. pneumonia + + + ++ + + ++ + _

P. mirabilis ++ + + ++ ++ + ++ ++ +

*++ (Heavy Growth of microorganisms), + (Moderate growth of microorganisms), - (No growth observed), a (MIC for S. typhi), b (MIC for S. typhi, S. dysentriae), c (MIC for E. coli, K. pneumoniae).

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4.7: Quantitative and qualitative analysis of total protein extracted from resistant bacterial isolates

Resistant bacterial isolates in disk diffusion method and well diffusion methods were sequestered for quantitative and qualitative analysis of protein. Protein extraction was done for a total of 12 samples by using protein extraction kit and isolated protein was analyzed on SDS-PAGE. 8%, 10% and 12% SDS-PAGE was done to optimize maximum band pattern analysis. Final SDS-PAGE was done on 10% resolving gel.

4.7.1: Bradford Macro assay

Concentration of unknown sample was calculated by following formula;

y = 0.0321 x -0.033

Where y = unknown sample absorbance;

X = unknown sample concentration.

Following Bradford assay for protein quantification, it was observed that unknown samples have total protein range from 1500 µg/ml to 2400 µg/ml for various bacterial isolates (Table 4.7.1).

Table 4.7.1. Absorbance and concentration of the Standards and the Samples at 595 nm. Label Absorbance (595nm) Concentration(µg/ml)

Standard sample 1 1.76 50

Standard sample 2 3.21 100

Standard sample 3 5.52 200

Standard sample 4 16.52 500

Standard sample 5 32.34 1000

Standard sample 6 63.87 2000 Unknown sample 1 46.77 1500

Unknown sample 2 65.48 2100

Unknown sample 3 74.83 2400 115

4.7.2: SDS-PAGE for total protein electrophoresis

10% SDS-PAGE was done for band pattern analysis of total protein extracted from various bacterial isolates. Final results of protein electrophoresis are shown in Fig. 4.7.2a and Fig. 4.7.2c. TotalLab Quant v11.5 provided the data based on the molecular density of each fraction for all isolates. Protein bands of varying molecular weight of control group were compared with resistant bacterial samples and all protein bands lied between 236.21 KDa – 10 KDa. All bands were found within the range of the protein ladder from 220 KDa to 10 KDa. Major heavier protein found in most of bacterial isolates had size about 230 KDa and other most common proteins bands lied in the range of >10, 14, 32, 80 and 200 KDa size (Fig. 4.7.2b and 4.7.2d) which exposed a considerablevariation in the cocncentration among different bacterial proteins. These total proteins were analyzed as a mixture of many proteins. In this study, total proteins of control samples lied mostly between 220, 130 and 30 KDa while total protein analysis of resistant strains revealed that proteins molecular weight of ≤10 KDa are noteworthy of observtion in results. 8 resistant bacterial strains were analyzed on SDS-PAGE and every strain showed different total protein fractions pattern depending on their physiology and cell wall structure (Table 4.7.2a-b). Observable protein fractions range of 231.73-9.63 KDa was noted for S. typhi whereas 235.65-8.4 KDa protein fractions were observed for K. pneumoniae. P. aeruginosa protein fractions were lying in the range of 227.82- 11.13 KDa and E. coli exhibit 222.70- 9.27 KDa on SDS-PAGE (Table 4.7.2a-b). S. aureus and P. mirabilis showed protein fractions ranging within 22 - 10.8 KDa and 219.52- 11.596 KDa, respectively (Table 4.7.2b). Thus, results of protein fractions resolved on SDS-PAGE in this study revealed that every bacterial isolate had different range of proteins that exhibit their different mechanism of resistance against antibiotics and disinfectants depending on structure of their cell wall and various physiological mechanisms inside the cell.

116

Figure 4.7.2a: 10 % SDS-PAGE gel 1 representing crude protein band patterns of resistant bacterial isolates of cockroaches. 117

Figure 4.7.2b: Lane-wise individual band molecular weight representation of SDS-PAGE gel 1.

118

Figure 4.7.2c: 10 % SDS-PAGE gel 2 representing crude protein band patterns of resistant bacterial isolates of cockroaches. 119

Figure 4.7.2d: Lane-wise individual band molecular weight representation of SDS-PAGE gel 2.

120

Table 4.7.2a. Lane-wise molecular weight description of bands of 10 %SDS-PAGE gel 1. Sr. No. Lane 1 kDa Lane 2 kDa Lane 3 kDa Lane 4 kDa Lane 5 kDa Lane 6 Lane 7 kDa Lane 8 kDa Lane 9 Lane 10 kDa kDa kDa

1 220000 227826.1 203386.1 225217.4 231739.1 227826.1 235652.2 217231.9 225333.3 236000 2 160000 199364.4 174325.7 204767.9 183273.5 143299.2 157757.7 161149.8 177252.9 214320.3 3 120000 135674 124503.1 174336.9 142351.9 103756.5 120000 110444.1 121241 118222.9 4 100000 96266.84 105834.5 129543.2 115593.8 91823.52 97218.26 87763.59 93542.37 84446.71 5 90000 82273.89 89087.42 112722.2 98120.27 82521.9 82463.18 77095.36 82173.67 67167.23 6 80000 62434.2 81239.38 93524.53 86809.72 76346.91 72993.71 68952.67 58089.78 37825.27 7 70000 52668.71 70692.35 83119.58 74893.92 56858.14 56616.33 56529.06 40135.84 30562.35 8 60000 42335.91 53172.98 77657.6 60230.95 41645.83 40000 42929.84 30112.25 23917.3 9 50000 30000 36281.41 66197.39 42514.39 36734.27 30218.97 38100.62 24250.14 20801.68 10 40000 22167.5 28226.32 46701.14 33821.59 30109.89 24802.57 32452.06 18007.75 13946.05 11 30000 15941.34 21699.42 35725.01 29335.16 24192.01 23192.67 28067.08 11997.93 11564.64 12 20000 10000 16067.78 26740.84 19887.57 16477.1 14215.74 22974.98 11448.12 8602.941 13 15000 14115.47 16950.21 16439.16 14636.47 11218.3 14732.14 9558.824 214320.3 14 10000 13383.83 13966.66 13966.66 11135.46 8478.261 12879.5 177252.9 118222.9 15 9637.681 11481.7 11615.1 9275.362

121

Table 4.7.2b. Lane-wise molecular weight description of bands of 10 %SDS-PAGE gel 2. Sr. No. Lane 1 kDa Lane 2 kDa Lane 3 kDa Lane 4 kDa Lane 5 kDa Lane 6 kDa Lane 7 kDa Lane 8 kDa Lane 9 kDa Lane 10 kDa 1 236216.2 229189.2 220000 222702.7 229189.2 220000 220000 105421.7 219531.7 153082 2 188774.8 183783.6 208564.1 196300.3 183783.6 160000 141229.2 63443.2 99810.9 67296.4 3 33975.6 73061.38 89481.18 88671.36 87618.54 120000 65612.57 51702 74015.4 49485.87 4 13476.16 37729.91 62440.39 61859.07 69427.98 100000 32443.25 40000 32004.45 37924.47 5 13368.89 13476.16 20000 47431.23 59099.01 90000 13476.16 35455.71 19211.66 15431.21 6 11363.18 13476.16 13476.16 25316.71 80000 10807.67 18720.6 13476.16 13476.16 7 10807.67 11205.75 13476.16 70000 13476.16 11596.94 11750.97 8 10967.58 60000 10807.67 9 50000 10 40000 11 30000 12 20000 13 15000 14 10000

122

Discussion

Although cockroaches contribute immensely to the transmission of bacteria, fungi and parasitic contaminants throughout the world, their role as vectors for transmission of infectious disease agents is generally ignored in all countries including Pakistan. The present study has revealed that cockroaches are capable of transmitting different infectious pathogens as various microbes have been isolated and identified form their body surfaces. Cockroaches are a common occurrence in human habitations particularly where food is stored, processed, prepared and served. Indeed, cockroaches’ are found everywhere and their omnivorous features makes them ideal carriers of pathogenic microorganisms.

Prior to applying any cockroach control measures, it is vital to undertake taxonomic work. In the present study, food-baited traps were installed in houses and hospitals in urban areas of Lahore to estimate year-around (4 trimesters) population dynamics of cockroaches. The identified species of cockroach species included P. americana, B. germanica, B. orientalis and B. lateralis (Table 4.1.2a). B. germanica as an indoor species at both sites (houses and hospitals) was the most abundant comprising 45% of total collections, followed by P. americana which was the second most abundant species in houses. Since distribution of cockroaches was observed indoors and outdoors throughout the year, it was evident from all the collection sites, the highest cockroach nymph’s collection (62%) was recorded in all traps followed by adult males (27%). It can be explained that as nymphs encountered with collection traps in search of food they found these as ideal places for inhabiting and future reproduction.

This pioneer study on year-round population estimate of cockroaches in Lahore, Pakistan revealed that cockroaches were more noticeable in traps in the 2nd trimester (July-September, 2013) most likely because average air temperature and relative humidity were most favorable for the breeding and development of cockroaches in this trimester (Table 4.1.2a). Relatively higher number of late instars were encountered in this trimester as they had gone through metamorphosis to change into adults and subsequently searching for their mates. However, during the first trimester (April-June, 2013) collection of early nymphal stages was more prominent as compared to adult males and females because that period was the vital season for breeding of eggs and development of early nymphal instars. 123

High prevalence of indoor species of cockroaches, including German and Turkestan cockroaches was observed in bedrooms, kitchen, stores, hospital wards, office stores and rooms adjacent to canteen (Table 4.1.2c) and their high abundance in these areas could be related to favorable shady, enriched food and cool environments conducive to their population growth. The outdoors cockroach species, American and Oriental cockroaches were found in the vicinity of sanitary pipes, washrooms, filthy habitats, adjacent gardens to houses, sewerage pipes and kitchen exit pipes where plenty of waste food was available (Table 4.1.2d).

The remarkable observation of this study was high infestation of B. germanica in hospitals and houses as compared to the offices, shopping malls/ departmental stores and institutes followed by P. americana infestation showing similar trends (Table 4.1.2c). Institutes and offices with basements and gardens were infested more with B. lateralis as compared to B. germanica. Houses (93.8%) were observed to be the most infested sites for cockroaches, followed by hospitals (89.5%) and offices (86.3%) where plenty of food sources and ideal shady places existed to support them. The least cockroach infested sites at dormitories of institutes/ universities (80%) and departmental stores (56.6%) could be explained due to proper implementation of pest control practices and relatively less waste food availability at these sites (Table 4.1.2c). Thus, indoor infestation rates of cockroaches was related to residential types; congested and more filthy habitats had high cockroach infestation rates in parricular. Findings of this study are in conformity with those of Majekodunmi et al. (2002) who had reported that houses were most commonly infested with B. germanica followed by apartments and villas. The intensity of cockroach infestation was related to the residential types.

Results of diversity indices for each cockroach species in all trimesters indicated lowest Shannon index of 1.1834 for 2nd trimester in which species abundance difference was more prominent compared to all other trimesters. Whereas, Simpson index was quite high in 2nd trimester (0.6547) compared to the 4th trimester (0.6687) revealing the observable change in species abundance for outdoors and indoors species (Table 4.1.2b). in the 4th trimester average air temperature was lowest and outdoors species of cockroaches were most prominently affected by change in temperature than indoors species. Lesser occurrence of outdoors species in traps can 124

be related to the drastic change in environmental temperature limitings the physiological and behavioral activities of cockroaches.

Relative abundance of cockroaches had positive correlation with the conditions of habitat especially seasonal fluctuation in temperature and humidity that affect life cycle and developmental stages of cockroaches (Table 4.1.2e). Most adapted outdoor cockroach species, P. americana was found to be the highest in all trimesters (Table 4.1.2d). Cockroaches were highly adapted for diverse land environment especially dry harsh environment. They were not much noticeable in cold months in third and fourth trimester but probably they have the capability of physical and behavioral adaptations which help them to withstand extreme low temperatures that prevailed in 3rd and 4th trimester. Environmental temperature plays an important role in population dynamics in a given habitat. Physiological adaptation of cockroaches to experience high or low temperature in their environment is compromised by heat shock proteins (HSP) in their cells which allow recovery on a cellular level (Lutterschmidt and Hutchison, 1997).

Previous studies have shown a positive correlation between the temperature sensitivity of many animals including cockroaches and their environmental temperature (Tsuji and Mizuno, 1973; Hu and Appel, 2004; Slabber et al., 2007; Appel et al., 2009). Results of the present study are in agreement with the findings of Snoddy and Appel (2008) who conducted a survey in Southern Alabama and Georgia, USA to determine the extent of B. asahinai and observed their bucket sample population increase in late August or early September.

Practice of personal protective measures and hygienic lifestyle have drastically reduced the prevalence of gastrointestinal infections, food-borne illnesses and nosocomial infections in our communities but other factors that contribute immensely to transmission of such infections are neglected. Speculations have always been made on the involvement of cockroaches as possible vectors of transmission of infective agents. Previously some studies in other countries including Iran, Iraq, Palestine, Ghana, Baghdad, India, Ethiopia, Taiwan, Brazil, Philippines, Thailand, Egypt, Nigeria, Algeria, Poland, Botswana and Morocco had been conducted to isolate pathogenic bacteria from cockroaches, however, no such work was undertaken in Pakistan that revealed the importance of cockroaches as possible transmission of 125

pathogenic bacteria, fungi and parasites residing on their external surface and digestive tract. Very little attention has been given to pathogenic importance of cockroaches in Pakistan.

In the present study, the two most active and abundant species of cockroaches, P. americana and B. germanica found in hospitals and houses in urban areas of Lahore were subjected to microbial screening, fungal and parasitic contaminants from external surface and digestive tract. Cockroaches were collected from 5 hospitals and 11 houses and every observed cockroach was infected with at least 1 or 2 types of bacteria so one can easily estimate the importance of cockroaches in possible transmission of pathogenic microorganisms especially bacteria. In this study 14 species of bacteria; E. coli, S. aureus, S. typhi, S. dysentriae, P. aeruginosa, S. epidermidis, P. mirabilis, P. vulgaris, E. aerogenes, S. pneumoniae, B. cereus, K. pneumonia, E. faecalis and E. cloacae were isolated from exterior and internal surfaces of cockroaches (Table 4.2.1b). The most common external isolate was E. coli (10.31%) followed by S. aureus (10.09%) but the most common external isolate was P. aeruginosa (19.96%) which is notorious for nosocomial infections followed by P. vulgaris (16.08%). S. typhi, S. epidermidis, E. aerogenes and S. pneumoniae were not isolated from gut tract of cockroaches (Table 4.2.1b). Absence of these bacteria in digestive tract of cockroaches can be attributed to the less compatible compartment for these bacteria as the rate of release for different bacteria with feces from digestive tract of cockroaches may vary. More gram-negative bacterial isolates were observed as compared to the gram-positive isolates from P. americana which indicated their possible entry route to human dwellings through sanitary pipes and filthy habitats.

Differences of external and internal bacterial isolates indicated that both surfaces have different pathological impact for their carriers. It can be explained by the enteric gram-negative bacteria being better adapted to invade the gut, where the stable and nutritious conditions are superior to those on the external surfaces of cockroaches. Human intestines are a reservoir for Proteus bacteria, especially those belonging to the most prevailing species, P. mirabilis and they are members of natural fecal microflora of large segment of the human population. Harmful strains of E. coli and S. typhi cause food poisoning in humans. Isolation of E. coli, S. typhi and P. vulgaris from the external surfaces of cockroaches revealed the possibility of their external 126

body surface contamination by these microbes during roaming in search of food and shelter. Female cockroaches were observed to be as high reservoir for S. aureus as compared to male cockroaches for both external and internal surfaces.

A major difference in the type and rate of infections can be related to the location of cockroach collection. Among hospitals, highest external bacterial infection for P. americana was observed in Punjab Institute of Cardiology (75.6%), while highest internal bacterial infection was observed in Sheikh Zayed Hospital (30.8%). Same trend was observed for B. germanica in which highest external bacterial contamination was found in Punjab Institute of Cardiology (32.4%) and highest intestinal bacterial contamination was observed in Sheikh Zayed Hospital (66%) (Table 4.2.2a). Most infected P. americana from Sheikh Zayed Hospital hospital and B. germanica from Punjab Institute of Cardiology hospital was due to their collection from an infected ward that was more infected than other hospitals. Existence of cockroaches in hospitals with highly infected pathogens, such as bacteria like Klebsiella and E. coli can cause bacterial epidemics in all wards from the infectious ward. Some people are allergic to excrement of cockroaches due to the presence of proteins in their feces. Hospital kitchens were more infested than medical wards and ICUs therefore; existence of cockroaches in hospitals can cause outbreaks of epidemics in different wards.

Among Houses, highest external bacterial infection for P. americana (55.9%) and B. germanica (52%) was observed for Shalamar town 2 locality. Whereas, highest internal contamination for P. americana (25.8%) was observed in Mughalpura 1 locality and for B. germanica (28.8%) was observed in Model town 3 houses (Table 4.2.2b). Shalamar town is included in newly developed Lahore city and has dense human population but poor infrastructure. Thus, poor sanitary facilities and highly populated areas with plenty of waste food sources make them ideal site for P. americana and B. germanica resulting in more efficient transmission of external bacterial contaminants. Location site of Mughalpura 1 and Model town 3 near canal bank road of Lahore with ideal humidity and temperature condition is conducive for cockroach encounter with disposable wastes and as a result make them ideal carrier for various pathogenic bacteria in their digestive tract. Bedrooms with attached washrooms and kitchens with open sanitary pipes were more infested with 127

cockroaches as compared to the stores/laundary rooms and bedrooms without attach washrooms. Faecal disposal facilities and sanitary conditions in houses also incurred in the prevalence of cockroaches as mechanical vector of bacteria.

In this study Shannon-Wiener’s diversity index was the highest for hospitals habitats (2.274) as compared to houses (2.167). Jaccard’s index of similarity for bacterial species found on cockroaches was smallest in the hospitals (0.281) indicating that bacterial species in the gut were different than those on the surface (Table 4.2.3). No correlation was observed between bacterial species isolated from external and internal surface of cockroaches in hospitals also suggests that the bacterial species in the gut exist somewhat independently relative to those on the surface (Table 4.2.1b).

Bray-Curtis index of dissimilarity for bacterial contaminants was lowest for three houses (0.0909) Shadman-2, Model town-1 and Johar town-1 indicating the same species composition for all three habitats (Table 4.2.3). Lowest Bray-Curtis index of dissimilarity indicated that the overall difference in bacterial composition was lowest and almost similar species were found in all collection sites. Shadman-2, Model town- 1 and Johar town-1 having similar residential types and infrastructure exhibited similar bacterial load for cockroaches collected from these sites.

Throughout the world scientists have worked on the isolation of E. coli, K. pneumoniae, S. aureus, P. aeruginosa, Shigella and Salmonella from cockroaches and have confirmed their presence (Islam et al., 2016; Solomon et al., 2016; Iboh et al., 2014; Isaac et al., 2014; Kassiri et al., 2014a; Menasria et al., 2014; Mikulak et al., 2013 and Tilahun et al., 2012). The present study also confirmed isolation of these pathogenic bacterial species; however isolation of S. epidermidis, P. mirabilis, P. vulgaris, E. aerogenes, S. pneumoniae, B. cereus, E. faecalis and E. cloacae of bacterial species is a new addition to the bacterial fauna isolated from external surfaces and digestive tract of cockroaches. Although few previous studies, e.g. Masood et al., 2014; Malik et al., 2013 and Mlso et al., 2005 had also reported E. coli, Salmonella, Staphylococcus spp. and streptococcus spp. from cockroaches in Pakistan. The present study contribute new knowledge about a large number of additional bacterial contaminants carried by cockroaches collected from indoors and outdoors areas of Lahore. 128

The number of areas containing waste refuse and human excreta provides best environment for population explosion of cockroaches and carriage of medically important bacteria. Hence, the relatively larger size of P. americana makes them a big source of pathogens carrying insects not only due to their size but also due to their association with more filthy habitats. The present study results indicate that all collected cockroaches from homes and even from hospitals harbored a large diversity of microorganisms. This abundance of microorganisms and specifically those resistant ones harbored in the body of the insect cause public health hazards, nosocomial infections and food-borne illnesses. Considering the significance of the present study, future studies on different population management methods against cockroaches should be carried out to control this menace.

Another important aspect of infections related to cockroaches is nosocomial fungal infection, an important cause of illness in children and immuno-compromised patients admitted in hospitals. Ventral abdominal crevices, thoracic joints and legs of cockroaches are typically ideal sites for attachment of nosocomial fungal spores. Food spoilage yeast and mycotoxigenic molds present on the external surface of trapped cockroaches specified them as an important reservoir of nosocomial fungi. The present study revealed that cockroaches represent a store of mycotoxigenic and spoilage molds that were isolated from external body surfaces of cockroaches and taxonomically identified by the First Fungal Culture bank of Pakistan. Since no previous work has been carried out in Pakistan concerning isolation of fungal contaminants from cockroaches, hence the present study is an addition to unveil the role of cockroaches in the transmission of various pathogenic agents including microbes and fungi.

In this study, cockroaches collected from different houses were found as reservoir of spoilage yeast including Geotrichum candidum (22%), Aspergillus flavus (75%), Metarhizium anisopoliae (20%), Metarhizium spp. (14%), Aspergillus oryzae (84%) and Aspergillus zonatus (50%) while mycotoxigenic molds included Alternaria alternate, A. niger, Mucor spp., Trichoderma spp. and Penicillium spp. (Table 4.3.1). These fungal isolates causing food poisoning, nosocomial infections are regarded as opportunistic pathogens. A. flavus has been known to produce mycotoxins which cause food poisoning in humans termed as mycotoxicosis. They may cause liver 129

damage and induce cancer in humans. Aspergillosis caused by Aspergillus fumigatus which attacks gut, lungs etc. and pulmonary aspergillosis is diagnosed as T.B. Geotrichum candidum caused an oral pulmonary, bronchial or intestinal infection in humans named as geotrichosis. Isolated Mucor spp. commonly found in soil act as cutaneous infectious pathogens among humans and also can grow on plant surfaces and rotten vegetables matters. Metarhizium anisopoliae also known as entomopathogenic fungus can produce mycotoxins that cause food poisoning in industries. Phytotoxins produced by Alternaria alternata cause pathogenicity in plants and play an important role in disease development.

The most prevalent fungal species isolated from cockroaches in the present study was Aspergillus oryzae (84%) which causes food poisoning by producing mycotoxins, followed by Aspergillus flavus (75%) and Apergillus zonatus (50%). Overall spores of Aspergillus spp. isolated from external surface of cockroaches were high in numbers. P. americana (85%) and B. germanica (53%) were contaminated by one or more species of fungal spores. Higher fungal contamination of P. americana can be attributed to their behavior, ecology and physiology. Being omnivorous and outdoors species they were encountering a variety of objects and as a result carried more fungal spores as compared to the B. germanica (Table 4.3.2).

P. americana and B. germanica collected from Shadman-1, Johar town-1 and Shalamar-2 were highly contaminated with fungal spores and showed relatively high prevalence. Environmental factors e.g. soil, air temperature and relative humidity have effect on the variety of fungal spores carried by cockroaches. While lowest prevalence of fungal contaminants for P. americana and B. germanica occurred in Shadman-2 and Mughalpura-2 (Table 4.3.2). Two collection sites Model town-1 and Johar town-2 showing similar carriage rate for both P. americana and B. germanica can be explained by their residential type and probability of encountering fungal spores. These two residential sites, with modern housing trend of covered lawn or no lawns which provide higher possibility for both species to share their habitat and carried same medically important fungal contaminants on their external surfaces.

In the past, there have been numerous reports on fungal contaminants of cockroaches in other countries but Pakistan, although it is an important public health issue. Motevali Haghi et al. (2014), Adeleke et al. (2012), Salehzadeh et al. (2007) and 130

Mpuchane et al. (2006) reported Candida spp., Schizosaccharomyces spp., and Zygosaccharomyces spp., Mucor spp., Rhizopus spp. and Trichoderma spp., Aspergillus flavus, Aspergillus ochraceus, Penicillium viridicatum, Penicillium variable and Fusarium graminearum). However, in the present study isolation of various fungal contaminants including Metarhizium anisopoliae, Aspergillus zonatus, Aspergillus oryzae, Geotrichum candidum and Alternaria alternate significantly contribute to the list of medically important fungal contaminants carried by cockroaches (Table 4.3.1).

Despite the abundance of cockroaches in urban area of Lahore and high prevalence of intestinal parasites in this urban setting, to our knowledge, there is no documented data on the role of cockroaches as carriers of intestinal parasites in urban area of Lahore. Lack of information on this important public health issue initiated this study and shed light on potential role of cockroaches for human parasitic transmission in urban settings. P. americana and B. germanica collected from different houses and hospitals were investigated for intestinal parasitic species of medical importance.

Present study results also indicated that 112 (44.8%) cockroaches harbored at least one human intestinal parasite on their body surfaces. The cockroaches from hospital environment (59.09%) harbored more parasites than houses (33.57%). Of the total, 76 (67.85%) cockroaches harbored parasitic protozoans and 36(32.15%) carried pathogenic and non-pathogenic intestinal parasites (Table 4.4.2a). P. americana harboured more parasites as compared to B. germanica in both environments (table 4.4.2b).

Common human intestinal parasites found in this study were A. lumbricoides, T. trichura, A. duodenalae, E. vermicularis, Taenia spp. and S. stercoralis, while the protozoans’ parasites included B. coli, E. histolytica, C. parvum, I. belli, G. duodenalis, E. coli and C. cayetenensis. Entamoeba coli (41.5%, 75.3%); Protozoan parasites were found as the most prevalent in both houses and hospitals followed by E. vermicularis (30.7%, 58.46%) and E. histolytica (30.7%, 61.5%). A. lumbricoides was relatively less prevalent in hospitals (52%) and houses (43%) for both species of cockroaches (Table 4.4.2a, Table 4.4.2b).

During the present study, both experimental sites were significantly different in carriage of parasitic load on cockroaches. Difference in the hygienic conditions of the 131

environments, including human excrement disposal, variable habitat interaction of, indoors and outdoors species, may account for the observed variation in the parasitic carriage rate of cockroaches among different experimental sites. Thus, findings of this study is that cockroaches are uniformly distributed in human habitation and act as a mechanical vector of pathogenic parasites that cause common illness, such as diarrhea and bowel disorders. This fact contributes to epidemiological chain; therefore, control of cockroaches will significantly lessen the prevalence of illness in human. Effective control strategies will reduce the public health burden of the gastro-intestinal parasites in the developing countries. This disparity in the number of parasites found in different habitat can be explained on the basis of the intense external contact with the parasites without ingesting them.

Previous studies conducted in different countries by Sia Su et al. (2016), Iboh et al. (2015b, 2014), Tilahun et al. (2012), Al-bayati et al. (2011), Al-Mayali et al. (2010), Kinfu and Erko (2008), Salehzadeh et al. (2007) reported high prevalence of parasitic contamination of cockroaches. In the present study, same parasitic species A. lumbricoides, T. trichura, A. duodenalae, E. vermicularis, Taenia spp. and S. stercoralis were isolated from external and internal surfaces of cockroaches collected from houses except for Entamoeba histolytica that were isolated only from external surfaces (Table 4.4.2b).

Another important contribution of this study was determination of antibiotic resistance patterns of bacterial isolates; results revealed high resistance rates observed against Ceftriaxone (CRO), Ciprofloxacin (CIP), Amoxicillin (AML), Cephradine (CE) and Tetracycline (TE) with the following order of preference of antibiotics AML > CE > TE > CRO > CIP. Highest resistance was observed against amoxicillin especially gram-positive bacteria exhibiting 100% resistance. Cephradine antibiotic was observed as second most resistant antimicrobial agent, followed by tetracycline as third most commonly observed antimicrobial agent. Ciprofloxacin was relatively strong antibiotic against most bacterial isolates. Although multiple antibiotic resistance and susceptibility varied among different antibiotics, majority of isolates showed high resistance to amoxicillin, followed by cephradine and tetracycline (Table 4.5a). Comparison of results of this study with disk diffusion supplemental tables 132

proposed by “CLSI, 2013” revealed higher resistance rate and prevalence of MDR isolates from cockroaches collected from houses and hospitals (Table 4.5a).

Furthermore, E. coli isolate exhibited 100% resistance to amoxicillin, cephradine and tetracycline but was sensitivity to ceftriaxone (68%) and ciprofloaxacin (88%)(Table 4.5a). However, S. aureus demonstrated 100% resistance to amoxicillin and resistance pattern for this microbe to ciprofloaxacin (80%), cephradine (53.3%), tetracycline (33.4%) and ceftriaxone (26%) was observed. Other isolates, S. typhi, and P. mirabilis, showed 100% resistance to amoxicillin while, P. aeruginosa exhibited 100% resistance to amoxicillin and tetracycline analogous to E. coli isolate of this study. Resistance patterns to various antibiotics were observed conflicting for S. dysentriae and K. pneumoniae. S. dysentriae was not completly resistant to any antibiotic but showed partial resistance to amoxicillin (50%), cephradine (40%), ceftriaxone (30%), ciprofloxacin and tetracycline (20%). K. pneumoniae exhibited 100% resistance to cephradine that was a great contradictory pattern as compared to other isolates while resistance profile for other antimicrobials was amoxicillin (70%), ceftriaxone (50%), tetracycline (40%) and ciprofloxacin (30%)(Table 4.5b).

In this study, highest sensitivity (80%) was observed to ciprofloxacin for all bacterial isolates, while S. typhi exhibited additional sensitivity to ceftriaxone (80%) and S. dysentriae exhibited additional sensitivity to tetracycline (80%) (Table 4.5a). Results also indicated that 6 bacterial isolates were resistant to 3 or more drugs suggesting the possible role of cockroaches as reservoir and vector of drug resistant E. coli, P. aeruginosa, S. aureus, S. typhi, P. mirabilis and K. pneumoniae in hospitals and this may contribute to nosocomial infections. Considering the fact that S. aureus is the most frequent cause of nosocomial infections caused by gram-positive bacteria, the detections of high number of multiple drug resistant isolates including those resistant to amoxicillin and cephradine, is a cause of concern as Amoxicillin is a penicillin antibiotic reserved for treatment of many different types of bacterial infections such as tonsillitis, bronchitis, pneumonia, gonorrhea, and infections of the ear, nose, throat, skin, or urinary tract, whereas cephradine belongs to cephalosporin antibiotics group and used to treat infections caused by bacteria, including upper respiratory infections, ear infections, skin infections, and urinary tract infections. 133

Results of this study proved that MDR strains had been demonstrated by cockroaches, however, MDR strains could arise due to accumulation of resistant genes in a single bacterial cell or expression of genes that code for multidrug efflux, extruding a wide range of drugs. Resistance to cephalosporins was due to extended spectrum beta lactamases (ESBLs) exhibited in gra- negative Enterobacteriacea. Significant aspect of this study was emphasizing on isolation of ESBLs presenting E. coli, K. pneumoniae and P. mirabilis pathogenicity and risk factors for transmission of gastrointestinal infections to humans. This study revealed that ciprofloxacin and ceftriaxone are effective antibiotics against Enterobacteriaceae while amoxicillin, cephradine and tetracycline should be used with precautions.

The high prevalence of MDR pathogens isolated from cockroaches in this study may be pointing to the fact that there has been a prolonged drug pressure of most of these antibiotics prescribed by doctors (Table 4.5b). High resistance to amoxicillin, cephradine and tetracycline may be due to their first choice antibiotic drugs in hospitals of Lahore perhaps because they are cheap and easily available. This misuse/abuse by general public may explain the continued resistance to antibiotics because of self-medication, over-dose and indiscriminate use of antibiotics in hospitals. The antibiotic resistance in pathogenic organisms had been reported to be plasmid mediated (Ariyo et al., 2015; Oleghe et al. 2011).

One of the major significances of the present study was to draw attention towards this important public health issue that surveillance on pattern and origin of MDR bacterial strains should not be limited to only clinical isolates as isolation of resistant strains from cockroaches proved them a possible potential vector. Observations made in the present study revealed that various bacterial isolates presented varying percentage for antibiotic resistance ranging from 20% to 100% (Table 4.5b).

Similarly, in the present study, patterns of resistance for cephradine, ciprofloxacin and ceftriaxone made significant contribution in the reported resistance patterns for microbial isolates of cockroaches and this information is being reported for the first time from Pakistan. However, 100% resistance to amoxicillin and tetracycline and low resistance for ciprofloxacin exhibited by S. aureus and E. coli in this study was in consistence with previous reports of Moges et al. (2016), Ghasemi-Dehkordi et al. 134

(2015), Brown and Alhassan (2014), Adeleke et al. (2012), Tilahun et al. (2012) and Kaleem et al. (2010).

However, in the present study regardless of geographical locations hospital’s environment is a reservoir for pathogenic microorganisms to a significant degree. Active use of disinfectants as biocides in cleaning and sanitizing different surfaces in hospitals is widely adopted all over the world. No previous study has been conducted in Pakistan on the consequence of commonly and frequently used disinfectants and biocides against the microorganisms isolated from cockroaches trapped from hospitals and houses. Therefore, the present study is an addition to the resistance profile of microbes isolated from cockroaches present in human dwellings. Estimation of biocide effectiveness of some disinfectants against the microorganisms isolated from cockroaches caught from hospitals and houses is another important contribution of this study. Germ kill vantocil IB {Poly (Hexamethylene biguanide) hydrochlorides (PHMB) 11%, G-cide as crystal HLD {Glutarol 9.8% vantocil GA and Germ Kill Vantocil FHC {Poly (Hexamethylene biguanide) hydrochloride (PHMB) 20% were tested by well diffusion method for 3 different dilutions of each disinfectant and inhibition zone diameter and MIC was assessed for each disinfectants against 7 bacterial isolates. Bactericidal effect of disinfectants depends on diffusion process of disinfectants in the media.

The present study revealed that all dilutions of Germ Kill Vantocil FHC disinfectant was more effective bactericide against all bacterial isolates particularly S. dysentriae, K. pneumoniae, P. aeruginosa and P. mirabilis. G-cide as crystal HLD was observed as 2nd most effective biocide against bacterial isolates of cockroaches, while Germ kill Vantocil IB was least effective bactericide. Germ kill Vantocil FHC exhibited the highest inhibition zone diameter for 12.5% diluton (27.5+11.23), for 25% dilution (24+11.05) and 14+13.856 at 50% dilution; similarly, RIZD was 81.81%, 54.54% and 4.54% for 12.5%, 25% and 50% dilutions, respectively (Table 4.6a). G-cide as crystal HLD showing inhibition zone diameter mean for 12.5% diluton (24+1.53), for 25% dilution (21.33+6.25) and highest inhibition zone diameter (26.66+4.61) at 50% dilution that is relatively lower than Germ Kill Vantocil FHC indicating its lower efficacy than RIZD 70%, 40% and 10% at 12.5%, 25% and 50% dilutions, respectively (Table 4.6a). While Germ Kill Vantocil IB showed inhibition zone 135

diameter mean for 12.5% diluton (22+3.06), for 25% dilution (17.33+2.40) and highest mean of inhibition zone diameter (22.11+1.68) at 50% dilution and recorded as the comparatiively lowest efficacy in all disinfectants applied against bacterial isolates of cockroaches with RIZD 45.45%, 18.18% and -9.18 at 12.5%, 25% and 50% dilutions respectively (Table 4.6a). The negative values of RIZD for any disinfectant indicated that its inhibition zone diameter was less than IZD for control group.

This study also revealed that biocide concentration of any disinfectant required to render effective cockroach control was higher than that recommended by the manufacturers and that sublethal application of control materials conducive to development of resistance may explain the high resistance profile of microbes isolated from the study cockroaches. Additionally, development of resistance may be due to wide and indiscriminate use and applications of disinfectants in hospitals by untrained staff. All the concentrated solutions of the disinfectants did not allow the growth of any bacteria. This may be partly due to the high concentration of the active ingredients in the undiluted portions and partly because they are not exposed to potential environmental contaminants.

MIC calculations for Germ kill Vantocil FHC for E. coli (Fig. 4.6b) and K. pneumonia was obtained at 50% dilution. S. aureus (Fig. 4.6a), S. typhi, S. dysentriae, P. aeruginosa and P. mirabilis indicated resistance to Germ Kill Vantocil FHC at 50% dilution. However, on the contrary, G-cide as crystal HLD exhibited MIC for S. typhi and S. dysentriae at 50% dilution and bactericidal activity was not observed for E. coli, S. aureus, P. aeruginosa and K. pneumonia but bacteriostatic activity was noticeable at 50% dilution of G-cide as crystal HLD. Germ kil Vantocil IB demonstrated MIC for only S. typhi at 50% dilution (Table 4.6b).

S. aureus, P. aeruginosa and P. mirabilis were found highly resistant to test disinfectants as bacteriostatic activity was observed for them instead of bactericidal action. While S. dysentriae showed the highest sensitivity in all isolates for all tested disinfectants and bacteriostatic as well as bactericidal activity of disinfectants was observed against S. dysentriae. K. pneumoniae exhibited higher sensitivity for all tested disinfectants and bactericidal results at 50% dilution of Germ Kill Vantocil FHC (Table 4.6b). 136

No previous study has reported the bactericidal activity of the disinfectants used in the presect study against microbes isolated from cockroaches inhabiting human dwellings. Therefore, no comparison of MIC %RIZD and effectiveness can be calculated for these disinfectants; however, few previous studies had reported antimicrobial efficiency of PHMB, Dettol and Phenol coefficient (PC) values of Polyhexamethylene biguanidine against gram-positive S. aureus and gram-negative K. pneumonia, P. aeruginosa and Klebsiella spp. by Okore et al. (2014), Al-Jailawi et al. (2015), Bouzada et al. (2010), Oule et al. (2008) Czajka et al. (2003), Mikulak et al. (2013) and Wallace (2001).

Most of the domestic areas in District Lahore including houses, hospitals, departmental stores, food handling establishments were polluted with domestic pollutants and provide ideal environment for breeding and multiplication of cockroaches. However, areas containing waste refuse and excreta of humans and domestic animals might provide best habitats for cockroaches; thus, pest control regulations, elimination of cockroaches from sensitive areas, such as hospitals, food- handling establishments and human dwellings should be adopted.

It is suggested that simultaneous use of bactericides and insecticides can reduce cockroach populations and bacterial densities in human dwellings. Although it is difficult to prove the involvement of cockroaches in direct transmission of pathogenic agents, isolation of pathogenic organisms that were of public health importance and medically important microbes carried by cockroaches in their body or on the outer surfaces should be noteworthy.

Development of model(s) for the evaluation of microbiological risks, related to cockroaches carrying microorganisms in and on their bodies in various habitats can be attempted. Cockroaches appear to be an important vector for multi-drug resistant neonatal nosocomial infections. In view of the existing threat of transmission of potentially pathogenic microorganisms, which show virulent features, and particularly those that are resistant to antibiotics and disinfectants, implementation of procedures to prevent infections in the hospitals and other cockroach inhabitation sites, including proper disinfection and insect control should greatly reduce the risk of occurrence of cockroach-related infections in habitats, such as hospitals and other sites where they co-exist with humans. 137

CONCLUSION

Cockroaches appear to be important vectors of disease agents by mechanical as well as biological routes. They act as potential transmitters of pathogens of nosocomial infections and food-borne illnesses in hospitals and house environments. The present study verified isolation of fungal and parasitic contaminants and MDR bacteria from cockroaches collected from different human dwellings. Development of resistance in bacteria due to intensive use of antibiotics is increasing day by day and warrants immediate attention to control this menace. A strong link between cockroaches and nosocomial infections was observed in the present study through isolation of bacterial pathogens that cause nosocomial infections from cockroaches trapped from hospitals and domestic dwellings. Cockroaches appeared to carry these pathogens on their legs, antennae and crevices of thorax and disseminate them on food and other places habitually. They may ingest bacteria by eating contaminated food and disseminate these pathogens after several days of ingestion. Control of cockroaches is an important phase in the strategic planning of preventing outbreaks of diseases caused by cockroaches. Essential sanitary and infrastructural improvements should be made in the architectural planning of buildings to mitigate breeding sites of cockroaches and eliminate their routes for inhabiting and movement within the buildings. Integrated Pest Management strategy should be adopted to control populations of cockroaches instead of a single chemical control method and this strategy should substantially reduce the spread of cockroach-related infectious diseases in our environment.

138

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APPENDICES

Appendix I. RECIPIE FOR TRYPTIC SOY AGAR (TSA)

Tryptone (Pancreatic digest of Casein) 15 gm

Soytone (Papaic digest of soybean meal) 5 gm

Sodium Chloride 5 gm

Agar 15 gm

pH = 7.3+0.2 at 25ºC

Appendix II. RECIPIE FOR MANNITOL SALT AGAR (MSA)

Proteose Peptone 10 gm

Beef Extract 1 gm

Sodium Chloride 75 gm

D-Mannitol 10 gm

Phenol Red 0.025 gm

Agar 15 m

Appendix III. RECIPIE FOR MACCONKEY AGAR

Peptic Digest of Animal Tissue 20 gm

Lactose 10 gm

Bile Salts 1.5 gm

Sodium Chloride 5 gm

Crystal Violet 0.001 gm

Neutral Red 0.05 gm

Agar 15 gm

Final pH 7.2 +0.2 at 25°C.

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Appendix IV. RECIPIE FOR EOSINE METHYLENE BLUE AGAR (EMB)

Peptic Digest of Animal Tissue 10 gm

Lactose 5 gm

Dipotassium Phosphate 2 gm

Sucrose 5 gm

Eosin-Y 0.40 gm

Methylene Blue 0.065 gm

Agar 13.50 gm

Final pH 7.2 +0.2 at 25°C.

Appendix V. RECIPIE FO Salmonella Shigella AGAR (SS AGAR)

Meat extract 5 gm

Peptone 5 gm

Lactose 10 gm

Ox bile, dehydrated 8.5 gm

Sodium citrate 10 gm

Sodium thiosulfate 8.5 gm

Ferric citrate 1 gm

Brillant green 0.0003 gm

Neutral red 0.025 gm

Agar 15 gm

Final pH 7.0 +0.2 at 25°C.

Appendix VI. RECIPIE FOR BLOOD AGAR PLATE (BAP) Meat extract 10 gm Peptone 10 gm Sodium Chloride 5 gm Agar 15 gm pH = 7.3+0.2 at 25ºC

Appendix VII. RECIPIE FOR SABOURAUD’S DEXTROSE AGAR

Mycological peptone 10 gm

Dextrose 40 gm

Agar 15 gm

pH adjusted at 5.6 at 25ºC

Appendix VIII. RECIPIE FOR MALT EXTRACT AGAR

Malt extract 30 gm

Mycological Peptone 5 gm

Agar 15 gm

pH adjusted at 5.4 at 25ºC

Appendix IX. RECIPIE FOR TRYPTIC SOY BROTH

Tryptone (Pancreatic Digest of Casein) 17 gm

Soytone (Peptic Digest of Soybean) 3 gm

Glucose (Dextrose) 2.5 gm

Sodium Chloride 5 gm

Dipotassium Phosphate 2.5 gm

pH adjusted at 7.3 + 0.2 at 25ºC

Appendix X. RECIPIE FOR MUELLER HINTON AGAR INGREDIENTS

Beef extract 2 gm

Acid hydrolysate of Casein 17.50 gm

Starch 1.50 gm

Agar 17 gm

pH adjusted at 7.3 + 0.1 at 25ºC

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Appendix XI. PREPARATION OF CRYSTAL VIOLET SOLUTION

Crystal Violet 20 gm

1% Ammonium Oxalate 80 ml

95% Methanol 200 ml

Dissolve 20 gm of crystal violet in 200 ml of 95% ethanol. Add this solution to 80 ml of a 1% Ammonium Oxalate solution. Let stand for 24 hrs and filter.

Appendix XII. PREPARATION OF GRAM IODINE SOLUTION

Iodine Crystals 3.33 gm

Potassium Iodide 6.67 gm

Add 3.33 gm Iodine crystals and 6.67gm potassium iodide to 1000 ml distilled water. Store in an amber bottle for further usage.

Appendix XIII. PREPARATION OF DECOLORIZER (95%)

Absolute Ethanol 95 ml

Distilled water 5 ml

Dilute 95 ml absolute ethanol to 100 ml with distilled water.

Appendix XIV. PREPARATION OF SAFRANINE SOLUTION

Safranin 2.5 gm

95% Ethanol 10 ml

Add 2.5 gm safranin to 10 ml 95% ethanol. Add this solution to 100 ml distilled water

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Appendix XV. OXIDASE TEST REAGENTS (1% KOVAC’S REAGENT)

tetramethyl-p- phenylenediamine dihydrochloride 0.1 gm

Distilled water 10 ml

It can auto-oxidize so use fresh reagents for each test. Strip of Whatman’s No. 1 filter paper were soaked in a freshly prepared 1% solution of tertramethyl-p-phenylene-diamine dihydrochloride. After draining for about 30 sec, the strips are freeze dried and stored in a dark bottle tightly sealed with a screw cap.

Appendix XVI. CATALASE TEST REAGENT

Commercially available 3% H2O2 Few drops on slide

Appendix XVII. COAGULASE TEST REAGENTS

Rabbit plasma obtained by centrifuging blood to which 0.1% EDTA has been added as anticoagulant. Stored the plasma in small portions at -20°C and in-used plasma at 4°C.

Appendix XVIII. INDOLE TEST REAGENTS (1% KOVAC’S REAGENT)

Tetramethyl-p-phenylenediamine dihydrochloride 0.1 gm

Distilled water 10 ml

It can auto-oxidize so always use fresh reagents not older than 1 week.

Appendix XIX. METHYLE RED VOGES PROSKAEUR (MRVP) MEDIA

Buffered peptone 7 gm

Glucose 5 gm

diPotassium Phosphate 5 gm

Dissolved in 1000ml of distilled water. Final pH 6.9 + 0.2 at 25ºC.

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Appendix XX. PREPARATION OF METHYL RED INDICATOR FOR MR TEST

methyl red 0.1 gm

95% ethyl alcohol 300 ml

Add sufficient purified water to make 500 ml. Store at 4-8ºC in a brown bottle.

Appendix XXI. PREPARATION OF BARRITT'S REAGENT FOR VP TEST

Solution A (Alpha-naphthol, 5% color intensifier)

α-naphtholin 6 gm

95% ethyl alcohol 100 ml

Solution B (Potassium hydrooxide, 40%, oxidizing agent)

potassium hydroxide 40 gm

Distilled water 100

Appendix XXII. SIMMONS CITRATE AGAR TEST REAGENTS

Sodium Chloride 5 gm

Magnesium Sulfate 0.2 gm

Sodium Citrate 2 gm

Bromo Thymol Blue 0.08 gm

Ammonium Dihydrogen Phosphate 1 gm

Agar 15 gm

Dipotassium Phosphate 1 gm

Distilled Water 1000 ml

Final pH 6.9 +0.2 at 25°C.

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Appendix XXIII. DISK DIFFUSION SUPPLEMENTAL TABLE APPROVED BY CLINICAL LABORATORY STANDARDS INSTITUTE (CLSI).

Antimicrobial agent Disc code potency Zone diameter (mm)

Resistant Intermediate Susceptible Ceftriaxone (CRO) 30 µg Enterobacteriaceae ≤19 20-22 ≥23 P. aeruginosa, Acinetobacter spp. ≤13 14-20 ≥21 and Staphylococcus spp. Ciprofloxacin (CIP) 5 µg P. aeruginosa, Acinetobacter spp., ≤15 16-20 ≥21 Staphylococcus spp. and ≤20 21-30 ≥31 Enterococcus spp. Enterobacteriaceae Amoxicillin (AML) 10 µg Enterobacteriaceae ≤13 14-17 ≥18 Staphylococcus spp. ≤19 - ≥20 Cephradine (CE) 30 µg Enterobacteriaceae, ≤14 15-17 ≥18 Staphylococcus spp. Tetracycline (TE) 30 µg Enterobacteriaceae and ≤11 12-14 ≥15 Acinetobacter spp. ≤14 15-18 ≥19 Staphylococcus spp., Enterococcus ≤24 25-27 ≥28 spp. and Vibrio cholera. S. pneumonia

Disk diffusion supplemental tables derived from “CLSI, 2013 document M100-S23 (M02-A11)”

Appendix XXIV. RECOMMENDED DOSE OF DISINFECTANTS BY MANUFACTURER

Disinfectant Recommended dose in water

Poly (Hexamethylene biguanide) 1.5 to 5 ml/L hydrochlorides (PHMB) 11%,

Glutarol 9.8%, 1.5 to 3 ml/L

Poly (Hexamethylene biguanide) 2 to 4 ml/L hydrochloride (PHMB) 20%,

Appendix XXV. BACTERIAL PROTEIN EXTRACTION KIT BS596 REAGENTS (BIO BASIC INC., CANADA).

Contents Quantity

10X cell lysis buffer 5 ml

DNase/RNase solution 200 µl

Lysozyme solution 1 ml

PMSF solution 500 µl

Appendix XXVI. PREPARATION OF 1 X PHOSPHATE BUFFER SALINE

10X PBS 50 ml

Double distilled Water 450 ml

Mixed well and stored at room temperature.

Appendix XXVII. PREPARATION OF 1X CELL LYSIS BUFFER

10X cell lysis buffer 200 µl

Double distilled Water 1800 µl

Eppendorf was rotated between palms to mix well and stored at 4ºC.

Appendix XXVIII. PREPARATION OF PHENYLMETHYLSULFONYL FLUORIDE (PMSF) SOLUTION

PMSF solution 0.8 µl

Isopropanol 79.2 µl

Mixed well and keep eppendorf at -20ºC for future use.

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Appendix XXIX. PREPARATION OF BRADFORD REAGENT

Coomassie Brilliant Blue G-250 50 mg

Methanol 50 ml

85% (w/v) phosphoric acid (H3PO4) 100 ml

Eppendorf was rotated between palms to mix well and stored at 4ºC.

Appendix XXX. 30% ACRYLAMIDE/ BISACRYLAMIDE SOLUTION

Acrylamide 29.2 gm

Bis-acrylamide 0.8 gm

Dissolve in 100 ml dd.H2O and stored at 4ºC.

Appendix XXXI. 1.5 M TRIS-HCL (pH 8.8) RESOLVING GEL BUFFER

Trizma base 18.16 gm

Make final volume upto 100 ml with dd.H2O and store at 4ºC.

Appendix XXXII. 1M TRIS-HCl (pH 6.8) STACKING GEL BUFFER

Trizma base 12.11 gm

Make final volume upto 100 ml with dd.H2O and store at 4ºC.

Appendix XXXIII. 10 SODIUM DODECYL SULPHATE SOLUTION (SDS)

SDS 10 gm

Water 100 ml

Store at room temperature.

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Appendix XXXIV. 10% AMMONIUM PER SULPHATE SOLUTION (APS)

APS 0.1 gm

Water 1 ml

Store at room temperature.

Appendix XXXV. 1X RUNNING BUFFER (SDS ELECTROPHORESIS BUFFER)

Tris Base 3.02 gm

Glycine 18.8 gm

10% SDS 10 ml

Make final volume upto 1000 ml and stored at 4ºC.

Appendix XXXVI. TRACKING DYE (LOADING DYE)

Dithiothreitol (DTT) 154 mg

SDS 200 mg

1M Tris (pH 6.8) 8 ml

Glycerol 10 ml

Bromophenol blue dye 20 mg

Thoroughly mixed up and covered eppendorf tube with aluminum foils and stored at 4ºC.

Appendix XXXVII. COOMASSIE STAIN (STAINING SOLUTION)

Coomassie blue R250 125 mg

Methanol 112.5 ml

Acetic acid 22.5 ml

Distilled water 112.5 ml

Dissolve completely and store in a dark bottle at room temperature.

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Appendix XXXVIII. COOMASSIE DESTAIN (DESTAINING SOLUTION)

Methanol 50 ml

Acetic acid 70 ml

Make final volume upto 1 litre with distilled water and store at room temperature.

Appendix XXXIX. BANDS AND CORRESPONDING MOLECULAR WEIGHTS OF PROTEIN LADDER

Band No. Molecular weight (KDa)

1 220

2 160

3 120

4 100

5 90

6 80

7 70

8 60

9 50

10 40

11 30

12 25

13 20

14 15

15 10

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Appendix XL. RECIPIE FOR 10% RESOLVING GEL

30% Acrylamide-Bisacrylamide 3.3 ml

1.5M Tris-HCl (pH 8.8) 2.5 ml

10% SDS 0.1 ml

Distilled water 4.0 ml

10% Ammonium per sulphate (APS) 100 µl

Mixed well and then added

TEMED (N-N-N’-N’-Tetramethylethylenediamine) 4 µl

(APS was always freshly prepared for each gel electrophoresis)

Appendix XLI. RECIPIE FOR 5% STACKING GEL PREPARATION

30% Acrylamide-Bisacrylamide 0.33 ml

1.5M Tris-HCl (pH 6.8) 0.25 ml

10% SDS 0.02 ml

Distilled water 1.4 ml

10% Ammonium per sulphate (APS) 20 µl

Mixed well and then added

TEMED (N-N-N’-N’-Tetramethylethylenediamine) 2 µl

(APS was always freshly prepared for each gel electrophoresis)