Double Disk Diffusion Test Research Articles

Prevalence of extended spectrum beta-lactamase producing Enterobacteriaceae in urine samples from Thumbay hospitals, U.A.E

Beta Lactamases is proven to be one of the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria. Many up to date researches have shown increase in the incidence and prevalence of ESBL worldwide. This study aimed to determine the prevalence of ESBL strains of Klebsiella spp. and Escherichia coli species in urinary isolates from the patients admitted in Thumbay hospitals around United Arab Emirates. Furthermore, drug resistant genes (SHV and CTX-M) in the ESBL positive samples were detected. 237 urine samples were collected from November 2017 to January 2018. Based on the lactose utilization, colony morphology, and biochemical utilization of the gram negative bacilli were identified as E. coli (53), Klebsiella pneumoniae (10) and Citrobacter species (2). Antibiotic sensitivity test, double disc diffusion test and combination disc tests all confirmed that the 65 (27.4 %) out of 237 isolates were ESBL producing bacteria. There was high prevalence of bacteria in females than male and the number of E. coli strains is higher than Klebsiella spp. DNA isolation was performed on the 65 samples, out of which 50 samples were selected for PCR based on their concentration. The selected DNA samples were used to detect the presence of bla CTX-M and bla SHV genes. Only 24 DNA samples (48 %) contains blaCTX-M genes, bla SHV or both the genes. 14 samples had bla CTX-M gene, 2 bla SHV genes, and 8 with both bla SHV and bla CTX-M. At the rate at which ESBL is spreading, further research, close observation and cautious use of antibiotics is important.

Microbiology quality, detection of enterotoxin genes and antimicrobial resistance of Staphylococcus aureus isolated from milk and Coalho cheese

This study evaluated the microbiological quality of milk and Coalho cheese, the prevalence of enterotoxin genes, antimicrobial resistance and determined an inducible MLSB resistance phenotype by the D-test in strains of Staphylococcus aureus isolated from these products. Seventy samples of milk and Coalho cheese were analyzed. S. aureus strains were identified by biochemical tests. The presence of se genes (sea-see) was tested by polymerase chain reaction. The antimicrobial sensitivity of S. aureus strains was evaluated for 13 antimicrobial drugs using the disk diffusion technique and the double-disk diffusion test (D-test) was performed to determine inducible resistance to lincosamide phenotype. The amount of toxin sufficient to cause foodborne diseases is generally observed when Staphylococcus populations exceed 105 CFU mL-1 g-1. In this study, none of the milk samples analyzed showed these counts; however, 73.3% (22/30) of Coalho cheese samples exceeded this value. A total of 109 isolates were identified as S. aureus. The presence of enterotoxin genes was detected in 25.7% of these isolates and amplified only for the sec gene. Most of the isolates (78.5%) were resistant to one or more antimicrobial agents. The D test showed that 25.0% of erythromycin-resistant isolates had the constitutive resistance phenotype, and 3.8% had the inducible resistance phenotype to clindamycin. These results indicate that these dairy products represent a health risk since these bacteria can cause foodborne diseases or may be a possible route for the transfer of antimicrobial resistance to humans.

Extended-spectrum β-lactamase producing E. coli in urinary tract infections: A two-center, cross-sectional study of prevalence, genotypes and risk factors in Amman, Jordan.

To determine the prevalence, phenotypes, and genotypes of extended spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) among patients with urinary tract infection along with identifying the associated risk factors. A cross-sectional study was conducted at two tertiary hospitals in Amman, Jordan between June and October, 2016. One hundred twenty one E. coli isolates from hospitalized patients with urinary tract infection were phenotypically assessed for ESBL production using the double disc diffusion test. Positive isolates to ESBL production were further genotyped using multiplex PCR. A nested case-control study was used to determine the independent risk factors. ESBL-producing E. coli were found in 75/121 (62%) isolates. Molecular genotyping demonstrated that CTX-M group1 (42.7%) predominated followed by combination of SHV and CTX-M group1 (20%). In the regression model, previous hospitalization and use of urinary catheter were identified as independent risk factors for ESBL-producing E. coli infections. We report a high prevalence of ESBL-producing E. coli which is in concordance with other studies from developing countries. Additionally, CTX-M group1 has emerged as the predominant ESBL produced by E. coli, which is consistent with reported results throughout the world. Independent risk factors to UTI infections due to ESBL-producing E. coli include previous hospitalization and use of urinary catheter.

Investigation of P Fimbriae Presence in Escherichia coli Strains Isolated from Urine Samples in Human, and Their Antibacterial Resistance

Background: In Turkey, Escherichia coli causing urinary tract infections (UTI) do not have current data on antibacterial resistance, extended spectrum β-lactamase (ESBL) enzymes types and virulence factors. Objectives: In this study, the isolation of E. coli from patients with UTI, determination of antimicrobial resistance of isolates, and investigation of the presence of ESBL enzymes and pyelonephritis-associated P fimbriae types, were aimed. Methods: Isolation E. coli from urine samples and identification were performed with conventional methods. Antibiotic resistance profiles were performed with the disc diffusion test (DDT). The presence of ESBL enzyme was investigated by the double disk diffusion test (DDDT). The presence of ESBL genes and P fimbriae genes were also investigated with PCR. The Chi squared test was used to compare the relationship between antibiotic susceptibility and resistance status in ESBL producing or non-producing and P fimbriae carrying and non-carrying isolates. Results: It was determined that 50.3% and 41.9% of isolates were resistant to ciprofloxacin, trimethoprim sulfamethoxazole. It was demonstrated that 49.7% of isolates were phenotypically ESBL positive, while 59.4% were genetically positive. The percentages of genes detected with PCR were 32.3%, 31.6%, 21.1%, 2.3% for blaTEM, blaCTX, blaOXA, blaSHV, respectively. A total of 51 pap genes were detected in 27.0% (36/133) of the total isolates. Conclusions: Extended spectrum β-lactamase producing E. coli infections associated with UTI increased in last years in Turkey. Further studies should be designed to investigate plasmids of ESBL positive E. coli stains isolated from UTI and whether plasmids are responsible for ESBL production.

Screening for ESBL producing bacterial isolates of agricultural soil and profiling for multidrug resistance

Abstract Objectives construction of agricultural environmental soil antibiotic resistance profile of the culturable bacteria in Kalyani, India, followed by the screening of ESBL producers harboring blatem, blashv, and blactx-M. Methods 158 Multi-Drug Resistant (MDR) bacteria were isolated from 18 agricultural sampling points in Kalyani, West Bengal, India followed by their Antibiotic resistance profiling against 15 test antibiotics. They were then screened for the presence of three Extended Spectrum Beta-Lactamase (ESBL) genes, namely blatem, blactx-M and blashv by Double Disk Diffusion test and Polymerase Chain Reaction. The positive candidates were further identified using Amplified Ribosomal DNA Restriction Analysis followed by 16S rDNA sequencing. Results lowest resistance was observed against Vancomycin (0%) whereas the highest against the β lactam Ampicillin (97.5%). Very high resistance was observed against the 3rd generation cephalosporin Cefotaxime (90%). 16/158 bacterial isolates were positive for one or more of the three ESBL genes under investigation. 11 out of those 16 isolates (68.75%) harbored at least 2 genes and the rest 31.25% either had the blashv or the blatem gene. Phylogenetic analysis of the prevalent ESBL carrying organisms in the region demonstrated that the genes are being horizontally transferred across different bacterial orders and classes. Conclusion Significant antimicrobial resistance noted against 12 out of 15 antibiotics tested. Stenotrophomonas, Myroides, Pseudomonas, Enterobacter, and Serratia are found as culturable MDR bacteria carrying ESBL genes among the isolates. It is the first report of environmental ESBL carriage by Myroides isolated from India.

PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF PSEUDOMONAS AERUGINOSA RECOVERED FROM KIDNEY LESIONS OF BROILER CHICKENS.

This work was planned to investigate the prevalence of P. aeruginosa in broiler chickens kidneys suffered from naked eye pathological lesions. Out of 240 examined kidneys samples, 25 isolates (10.4%) of P. aeruginosa were recovered. Antimicrobial sensitivity pattern against 14 different antimicrobials comprising nine groups proved that isolates were resistant to most of the used antimicrobial agents. Phenotypic detection of extended spectrum beta-lactamases (ESBLs) producing isolates using modified double disk diffusion test revealed that, ten (40%) were ESBLs producers. Genotypically using PCR, three ESBLs genes (TEM, Ctx-M and Shv) and three virulence genes (lasI, Tox A and Plc H) were detected with different percentages. Cinnamon and carvacrol oils were found bactericidal for P. aeruginosa at concentrations of 4% and 0.4% respectively.

Detection and Molecular Typing of Extended Spectrum Beta Lactamases (ESBLs) Producing Gram-Negative Bacteria from Wound Infections

ABSTRACT: Extended spectrum β-lactamases (ESBLs) producing bacteria, which are mainly Gram-negative, have emerged as a major threat in recent years as they are resistant to most antimicrobials. This study was conducted to investigate the prevalence of ESBL producing Gram-negative bacteria in woundinfections and to map their antimicrobial resistance profile. A total of 100 samples were collected and identified from infected wounds. The Gram-negative bacteria were checked for identification as ESBL producers by using double disc diffusion test. The identified ESBL producers were subjected to PCR basedtyping of ESBLs. Out of 100 collected infected wound samples, 69 Gram-negativebacteria were isolated. The isolated pathogens were: Klebsiella pneumoniae (45%), Escherichia coli(29%), Enterobacter(10%), Proteus spp (9%) and Pseudomonas aeruginosa(7%).Twenty eight (40%) isolates were detected as ESBLproducers The ESBL producers were subjected to molecular analysis that showed CTX-M (82%) as most prevalent enzyme type responsible for ESBL production. One E. coli and one Proteus isolate was identified as GES and PER type ESBL producer respectively, where as one of the E. coli isolates was found to harborboth CTX-M and GES type ESBLs. TEM and OXA types were not found in any of the isolates. All ESBL producers showed multidrug resistance for eleven antimicrobials. Klebsiella (40%) was identified as the most prevalent ESBL producer followed by E. coli (35%). Most dominant ESBL type identified by PCR wasCTX-M and Klebseilla pneumoniae (83.3%) and E. coli (100%) were the dominant CTX-M type ESBL producers.

Ceftazidime-avibactam and Meropenem Double Disk Diffusion Test for Identifying Carbapanem-Resistant Enterobacteriaceae and Distinguishing Between Serine and Metallo-β-Lactamase Producing Organisms

BackgroundEarly detection of carbapenem-resistant Enterobacteriaceae(CRE) is crucial for selection of effective treatment. While KPC is the most prevalent carbapenemase in the US, phenotypic screening methods, such as the carbapenemase inactivation method (CIM) and CarbaNP, cannot easily distinguish between serine and metallo-β-lactamases (MBL). The aim of this study was to evaluate a simple double disk diffusion (DD) test to confirm carbapenem (meropenem) resistance (MER disk) and that resistance was due to a serine carbapenemase as indicated by susceptibility to ceftazidime-avibactam (CAZ-AVI disk). MBL-producing organisms are likely to be resistant to both MER and CAZ-AVI.MethodsIn total, 83 clinical isolates of Enterobacteriaceae were selected for the validation: 54 Klebsiella pneumoniae (KP), 16 Enterobacter cloacae (ECL) and 13 Escherichia coli (EC). All isolates were screened for specific β-lactamase genes (Checkpoints, Wageningen, Netherlands) and included KPC, OXA, IMP, VIM, NDM as well as strains with KPC and alterations on OmpK35 and OmpK36. Isolates were tested for susceptibility to MER and CAZ-AVI by disk diffusion and broth microdilution (BMD) per CLSI guidelines. Results were analyzed to evaluate suitability of the DD test to distinguish between serine and MBL-producing organisms.ResultsOverall correlation between disk and BMD was 97–100% for CAZ-AVI and 94–100% for MER. Among the 50 CRE that were susceptible to CAZ-AVI were strains positive for KPC, or OXA, or in combination with ESBLs. Among the 16 isolates that were resistant to both CAZ-AVI and MER were strains that produced MBLs such as IMP, VIM and NDM and included strains with alteration in OmpK35 and OmpK36. Among the 17 carbapenem-susceptible control strains all were susceptible to both agents and were positive for AmpC or ESBLs.ConclusionThe CAZ-AVI and MER DD test was successful in confirming CRE phenotype and in distinguishing between serine carbapenemase-producing and MBL-producing organisms. The test will be useful in screening patients in future trials to evaluate the efficacy of CAZ-AVI in global CRE studies where MBL’s are more prevalent in other geographic regions. Both disks are commercially available and can be performed in most clinical laboratories.Disclosures L. Y. Lin, Allergan plc: Employee, Salary; I. Critchley, Allergan plc: Employee, Salary; D. Melnick, Allergan plc: Employee, Salary

Occurrence of extended-spectrum β-lactamase, AmpC and virulence genes in Escherichia coli isolates from vegetable salads in Morocco

PurposeThe purpose of this paper is to assess the occurrence of the extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamase genes in 144 Escherichia coli isolates recovered from 160 vegetable salad samples.Design/methodology/approachAmong the 144 E. coli isolates recovered from 160 vegetable salads, 17 (12 percent) ceftazidime-resistant isolates were screened for ESBL production with the double disk-diffusion test. The ESBL-producing isolates were characterized for antimicrobial resistance, the presence of virulence genes and plasmid-mediated quinolone resistance (PMQR) determinants. The isolates were also subjected to phylogenetic group typing. The existence of plasmid AmpC genes and mutations in the regulatory region of the chromosomal AmpC gene was assessed using polymerase chain reaction (PCR) and sequencing. All β-lactamase isolates were further characterized by pulsed-field gel electrophoresis to determine the genetic relatedness.FindingsOverall, 17 (12 percent) of the 144 E. coli isolates studied were ceftazidime resistant. Among the 17 isolates, 13 (77 percent) were multidrug resistant and four (23.5 percent) were ESBL producers. The bla CTX-M14 was the only gene detected. Of the 12 AmpC-producing isolates, three (18 percent) harbored plasmid-encoded AmpC and sequencing analysis of the chromosomal AmpC genes revealed mutations in the promoter/attenuator region. PMQR determinants were detected in 9 (52 percent) isolates. A was the most prevalent phylogenetic group (56 percent), followed by groups B1 (31 percent), D (6 percent), and B2 (6 percent). PCR showed that six (50 percent) ESBL/AmpC-producing E. coli isolates carried one and/or two virulence genes. Pulsed-field gel electrophoresis showed no epidemiological relationship between these isolates.Originality/valueThis study places vegetable salads within the spectrum of ecological niches that may be vehicles for antibiotic-resistant bacteria/genes with clinical interest and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.

Biochemical, Virulence and Resistance Features in Bacterial Strains Recovered from Hospital Surfaces after Decontamination with Quaternary Ammonium Compounds, Triclosan and Iodine Desinfectatnts

The purpose of this study was to investigate the resistance and virulence markers in microbial strains isolated from the hospital environment, recovered after surfaces decontamination with quaternary ammonium compounds, triclosan, iodine disinfectants, in order to predict their role in healthcare-associated infections. The resistance phenotypes were established using disk diffusion and double-disk diffusion test. Simplex and multiplex PCR assays were used to identify the genetic support of cell-associated and soluble virulence factors The Gram-negative bacterial strains isolated from hospital surfaces after decontamination were resistant in high proportions to beta-lactam antibiotics, including penicillins and associations with beta-lactamase inhibitors, third and fourth generation cephalosporins and carbapenems (encoded by blaTEM, blaNDMlike blaCTX-Mlike, blaOXA-48like genes), quinolones (QnrA, gyrB, parE genes), aminoglycosides (aac3Ia gene), and tetracyclines. Regarding the virulence profiles, P. aeruginosa strains revealed the plcH gene (86.6%) followed by plcH, ExoS, algD and ExoU, while the Enterobacteriaceae strains, fimH (24%), followed by papC, sfaDE, hlyA, cnf1, eaea and VT2 genes. S. aureus isolates revealed the SCCmec cassette of type II (40%), followed by type V, IIIA and type IV (2B) and the clfA, clfB, and can adhesion genes. These findings demonstrate that incorrect practices in biocides use, regarding contact time and concentrations or instruments and surfaces insufficient cleaning before disinfection can decrease their effect and favor the persistence of resistant and virulent microbial strains in the hospital environment. The multiple drug resistance and virulence determinants, encoded by diverse genetic elements suggest the potential of these strains to persist and initiate hospital-associated infections.

Erm gene distribution among Norwegian Bacteroides isolates and evaluation of phenotypic tests to detect inducible clindamycin resistance in Bacteroides species

The aims of this study were to describe the distribution of the most common erm genes in a collection of Norwegian Bacteroides isolates and to investigate whether the phenotypic tests for determining inducible clindamycin resistance among Bacteroides species recommended by EUCAST, NordicAST and the manufacturer of E-test®, are effective.We investigated 175 unique Bacteroides isolates for the presence of erm(B), erm(F) and erm(G) genes, determined their minimum inhibitory concentrations (MICs) to clindamycin and categorised their susceptibility according to EUCAST breakpoints. 27 isolates were resistant to clindamycin. Furthermore, we investigated whether these recommended methods could detect inducible resistance in the Bacteroides isolates: 1) EUCAST recommendation: Dissociated resistance to erythromycin (clindamycin susceptible with erythromycin MIC > 32 mg/L), 2) NordicAST recommendation: Double disk diffusion test (DDD) or 3) Manufacturer of E-test®’s recommendation: prolonged incubation of clindamycin E-test® for 48 h.erm genes were detected in 30 (17%, 95% CI 12%–23%) of 175 Bacteroides isolates with erm(F) as the dominating gene. There were six (4%, 95% CI 1%–7%) of 148 clindamycin susceptible isolates harbouring erm genes, they were considered inducibly resistant to clindamycin. None of the methods for phenotypic detection of inducible clindamycin resistance performed satisfactory with sensitivities of 33%, 17% and 0% and specificities of 90%, 99% and 97% for dissociated resistance, DDD and prolonged incubation of clindamycin E-test®, respectively.In our view, the scientific basis for investigating every Bacteroides isolate for inducible resistance to clindamycin is weak. Molecular detection of erm genes may prove a better option than the phenotypic methods we evaluated.

Quinolone Resistance Determinants qnr, qep, and aac(6’)-Ib-cr in Extended-Spectrum Β-Lactamase Producing Escherichia coli Isolated From Urinary Tract Infections in Tehran, Iran

Background: Prevalence of plasmid mediated quinolone resistance in Escherichia coli clinical isolates is a serious problem in developing countries. Objectives: The aim of this study was to investigate quinolone resistance determinants among Extended-Spectrum Β-Lactamases (ESBL)-producing E. coli recovered from patients with nosocomial urinary tract infection. Methods: A total of 290 E. coli isolates, obtained from patients with UTI, were included in this study. The phenotypic confirmatory test for ESBL production was performed by double disc synergy test and combined disk diffusion test. Kirby-Bauer Disk diffusion method was performed to test susceptibilities of ESBL-producing E. coli isolates to 15 antimicrobial agents. Isolates were screened for the presence of ESBL and plasmid-mediated quinolone resistance genes by polymerase chain reaction (PCR). Results: In the present study, 290 (71.7%) E. coli strains were recovered from 410 hospitalized patients with UTI, 51.7% of which were found to have ESBL positive results. In vitro, the susceptibilities of ESBL-producing E. coli strains showed that all isolates were resistant to amoxicillin and penicillin and resistance rates to other of antibiotics differed from 40% to 96%. Among the 150 ESBL positive isolates, frequencies of aac(6’)-Ib, oqxA, oqxB, qnrA, qnrB, qnrS, and qepA were 74.7%, 8%, 4%, 3.3%, 1.3%, 2%, and 2.7%, respectively. Coexistence of bla CTX-M, bla TEM, bla CTX-M, and aac(6’)-Ib were the most widely distributed resistance genotypes. Conclusions: The data of the present study revealed the high prevalence of plasmid-mediated quinolone resistance genes among ESBL-producing E. coli in the hospitals. The bla CTX-M genes were found to be the dominant ESBL-encoding gene.

Urinary Tract Infections in Patients with Type 1 and Type 2 Diabetes: Etiology, Resistance to Antibacterial Chemicals and Virulence Features

One of the most frequent chronic complications occurred in diabetes patients are the urinary tract infections (UTIs). This study aimed to investigate the incidence of UTIs in a cohort of 93 (47 males: 46 females) diabetic patients, the prevalence of different microbial species involved and their virulence and antibiotic resistance profiles. The identification of the uropathogenic strains in the positive urine samples was performed using conventional methods and API tests. After identification, the antibiotic susceptibility profiles were established by the standardized disk diffusion method and double disk diffusion test was performed for the confirmation of ESBL and inducible AmpC b �lactamase phenotypes. The isolated strains were tested for the production of different cell associated and soluble virulence factors, i.e.: bacterial adherence to cellular substrata (HeLa cells), hemolysins (hemolysis spot, CAMP-like), amylase, caseinase, aesculin hydrolysis, DNA-ase, lipase and lecithinase. In the analyzed group, the total prevalence of UTIs was of 46%, a higher incidence being observed in the female patients (64%). Similar to other studies, the etiology of UTI in the investigated diabetes patients was dominated by E. coli, followed by Klebsiella sp. strains. The isolated strains preserved good susceptibility rates to quinolones and aminoglycosides and revealed important virulence features, related to their capacity to colonize the cellular substratum and to produce soluble virulence factors involved in persistence, colonization and progression of the infectious process. The high percentage of beta-lactam resistant strains (including carbapenem-resistant ones) requires careful surveillance of the dynamics of susceptibility profiles for limiting the emergence of these strains in community.

English

Widespread use of Macrolide, lincosamide and Streptogramin B (MLSB) family of antibiotics in the treatment of Staphylococcus aureus (S. aureus) has led to an increased resistance to MLSB antibiotics. The purpose of this study was to determine the frequency of MLSB resistance among clinical isolates of methicillin sensitive S. aureus (MSSA) and Methicillin resistant S. aureus (MRSA) from Mansoura University Children Hospital (MUCH), Egypt, phenotypically by using D‑test and genotypically by detection of erm genes by PCR. Different microbiological samples were collected under complete aseptic condition from patients in MUCH according to the site of infection over a period of 9 months from March 2016 to November 2016. S. aureus isolates were identified using standard microbiological methods. MRSA was detected by growth on oxacillin screen agar plate and cefoxitin disk screen test. Antimicrobial susceptibility of the isolates was determined by Kirby-Bauer disk diffusion method. S. aureus isolates that were found to be erythromycin resistant were further studied for inducible clindamycin resistance using D-zone test according to CLSI recommendations. erm genes in S. aureus isolates were detected by PCR. Among 230 S. aureus isolates, 164 were MSSA (71.3%) and 66 were MRSA (28.7%). Twenty-five MSSA (15.2%), and 37 MRSA (56.1%) isolates were erythromycin resistant. Constitutive MLSB phenotype (cMLSB) (30.3 and 4.2%) and inducible MLSB phenotype (iMLSB) (22.7 and 7.9%) were observed in MRSA and MSSA, respectively by D-zone test. The rate of iMLSB phenotype and cMLSB phenotype in MRSA was significantly higher than in MSSA isolates. The frequency of ermA, ermB and ermC genes were 72.9, 5.4 and 13.5% in MRSA isolates and 60, 4 and 12% in MSSA isolates, respectively. Screening test for of iMLSB‑resistant strains is very important by double disk diffusion test (D‑test). This phenotypic test is simple, accessible and reliable method that can be done in every laboratory and research facility, without the need of costly genetic tests. Since the treatment of patients infected with S. aureus with iMLSB phenotype with clindamycin can lead to the expansion of constitutive resistance (cMLSB) and therapy failure. Key words: Clindamycin, cMLSB, erm genes, iMLSB phenotype, MRSA, MSSA, S. aureus.

ESBL Positive Organisms: Method of Routine Reporting and Prevalence in Health Care Settings

Extended Spectrum ß- Lactamase (ESBL) acquired by bacteria is a highly effective weapon for drug resistance against a wide range of antibiotics (penicillins, cephalosporins and monobactams). ESBLs have continued to increase in variety and prevalence and are now a global health concern including Bangladesh. They are associated with failure in effective treatment, increased morbidity and mortality, poor outcomes, increases in length of stay (LOS) in hospital and health care costs. So, it is important to identify it and take necessary measures to treat with appropriate antimicrobial therapy. This study has been designed to establish an easy method for routine reporting of ESBL organisms and notify it's incidence in Health Care settings. Double Disk Diffusion Test, utilizing Cefotaxime, Amoxicillin/Clavulanic acid and Ceftazidime, was used as Screening Test. Phenotypic Confirmatory Test was done for confirmation of screening ESBL positive organisms. Four hundred seventy two organisms (Escherichia coli and Klebsiella sp.) were isolated from urine and pus collected from Health Care settings. Only 50 screening ESBL positive organisms and 50 ESBL negative organisms were tested by Phenotypic Confirmatory Test. All of them were positive and negative respectively by Phenotypic Confirmatory Test. Predominant organism among the isolates was Escherichia coli (82.8%) of which 54.4% were ESBL positive followed by Klebsiella sp. (17.2%) of which 44.4% were ESBL positive. ESBL positivity was more in hospital infections (73.3%) than in community acquiredinfections (44.5%).Bangladesh J Med Microbiol 2014; 08 (02): 23-27

Prevalence and Molecular Characterization of Extended Spectrum Beta Lactamases Producing Escherichia Coli Isolates Causing Hospital - Acquired and Community - Acquired Urinary Tract Infections in Sohag University Hospitals, Egypt

Background: Escherichia coli (E. coli) are the most common etiological agents of hospital acquired and community acquired urinary tract infections (UTIs) and the incidence of antibiotic resistant strains has been steadily increasing over the past few years resulting in limitation of therapeutic options. Our objectives were to investigate the prevalence and molecular characterization of extended spectrum β- lactamases (ESBLs) producing E. coli isolated from out-patients and hospitalized patients with UTI attending Urology Department of Sohag University Hospitals during the period from May 2014 to May 2015. Methodology: Forty four E. coli isolates were collected during the study period. The isolates were tested for antibiotic sensitivity by the disc diffusion method. All isolates were screened phenotypically for ESBL production by the double disc diffusion (DDD) test. The presence of bla CTX-M-I, bla TEM and bla SHV genes in ESBL- producing E. coli was tested by polymerase chain reaction (PCR). Results: showed high prevalence of ESBL-producing E. coli; as 88.6% of the collected isolates were ESBL producers. Twenty- eight (71.8 %) isolates were multidrug resistant (MDR). CTX-M-I gene was the most prevalent ESBL type (95 %); followed by TEM (53.8%) and lastly SHV gene which was detected only in 10.3% of cases. Conclusions: E. coli isolates carrying multiple ESBL genes are widespread in our locality. CTX-M-I -producing E. coli are currently a problem which may be related to the misuse of third generation cephalosporins, especially cefotaxime. Continuous monitoring, effective infection control measures and judicious antibiotic usage are required.

Survey of strain distribution and antibiotic resistance pattern of group B streptococci (Streptococcus agalactiae) isolated from clinical specimens.

Aim: The aims of the present study were to determine the antibiotic susceptibility profils with particular emphasis on susceptible or resistant strains to macrolides and lincosamids antibiotics and to determine possible antibiotic resistance mechanisms occurring in group B streptococci (GBS) strains using PCR assay and disk diffusion method.Methods: A total of 62 clinical GBS strains were investigated. Antibacterial susceptibility testing was performed using the disk diffusion method and inducible resistance test for clindamycin by standard double disk diffusion or D-zone test for all isolates to differentiate macrolide resistance phenotype (M), constitutive macrolide-lincosamide-streptogramin B phenotype (cMLSB) and induced macrolide-lincosamide-streptogramin B phenotype (iMLSB). In addition, minimum inhibitory concentrations (MIC) of penicillin were determined for all isolates. Finally, possible existence of antibiotic resistance genes for erythromycin (ermTR, ermB and mefA/E) and for clindamycin (linB) were examined among isolates using PCR assay.Results: All 62 isolates were susceptible to penicillin, ampicillin, linezolid, cefazoline and vancomycin. However, 93.5% (n=58) of isolates showed an increased MIC to penicillin. The overall rate of erythromycin resistance was 35.5% (n=22). All erythromycin-resistant isolates displayed the M phenotype (100%, n=22). All three erythromycin resistance genes (i.e. ermTR, ermB and mefA/E) were found in erythromycin-resistant isolates.Conclusion: It was concluded that prescribing antibiotic without antibacterial susceptibility tests should be prevented because of the high prevalence of erythromycin-resistant GBS strains and the fact that erythromycin-resistant GBS strains has shown an increased MIC to penicillin, as the drug of choice for treating GBS infections.

Prevalence of ESBL-mediated Resistance among Hospital and Community isolates of Klebsiella pneumoniae in Warangal

Introduction: Extended Spectrum β-Lactamase (ESBL) producing Klebsiella pneumoniae were prevalent in the hospital environment and were responsible for many hospital associated infections (HAIs).The present study was aimed at identifying these strains in the hospital environment so as to guide the clinician in planning antibiotic policy. Material and Methods: 200 clinical isolates of Klebsiella pneumoniae constituted the study group.100 isolates were from the community and 100 from the hospital. Based on their susceptibility or resistance to 3rd generation cephalosporins (3GCs), the isolates were characterized into two groups. Group I consisted of sensitive strains(128 numbers) which showed a zone diameter of more than 17mm and group II consisted of resistant strains(72 numbers) which showed a zone diameter of less than 17mm to ceftazidime (30µg), cefotaxime (30µg) and ceftriaxone (30µg). Isolates of group II were tested for ESBL production using two methods namely double disc synergy test (DDST) and phenotypic confirmatory disc diffusion test (PCDDT). Results: Among the 72 resistant strains, 36 (50%) were ESBL producers and 36 (50%) were non-ESBL producers. Among the hospital isolates, the percentage of ESBL producers was more (30%) compared to the community isolates (6%).Conclusion: About one third of the hospital isolates of Klebsiella pneumoniae showed ESBL production whereas ESBL mediated resistance was low in community isolates. This study substantiated the need for planning proper antibiotic policy to reduce mortality due to Gram-negative sepsis.

Molecular characterisation of Klebsiella oxytoca strains isolated from patients with antibiotic-associated diarrhoea

Background and study aimColitis is a common complication after treatment with antibiotics such as β-lactams, quinolones, and aminoglycosides. Recently, Klebsiella oxytoca has been implicated in this type of diarrhoea. The prevalence and characterisations of K. oxytoca isolated from patients with antibiotic-associated diarrhoea were investigated. The K. oxytoca isolates were also tested for cytotoxin production. Patients and methodsThis study was conducted from May 2011 to Dec 2013. Faecal samples were collected from hospitalised patients receiving antibiotic treatment. Initial cultivation was performed on specific media. The clinical isolates were confirmed by polymerase chain reaction (PCR) using the specific K. oxytoca polygalacturonase (pehX) gene. The double-disc diffusion test was used to detect extended-spectrum beta-lactamase (ESBL)-producing strains. Tracking of ESBL-encoding genes was performed via PCR. The organism was cultured on Hep-2 cell lines for cytotoxin production. ResultsOut of 331 samples collected from patients, 40 were confirmed molecularly to be clinical isolates of K. oxytoca. Fourteen (35%) ESBL-producing strains were isolated using the double-disc diffusion method. Among the molecularly confirmed K. oxytoca isolates, seven (17.5%) tested positive for the blaSHV gene, 12 (30%) for blaTEM, 10 (25%) for blaCTX-M, three (7.5%) for blaOXA, nine (22.5%) for blaCTX-M-15, and seven (17.5%) for blaTEM-1. Five (12%) isolates showed cytotoxin activity below 30%, 12 (30%) strains showed moderate cytotoxin activity between 30% and 60%, and 23 (58%) strains showed cytotoxin activity ⩾60%. ConclusionsThe cytotoxin-producing K. oxytoca is found to be one of the causes of antibiotic-induced colitis. Discontinuing treatment and allowing normal intestinal flora to be established or prescribing appropriate medication after antibiogram can help patients with antibiotic-induced haemorrhagic colitis in a timely manner.

Healthcare-Associated Methicillin-Resistant Staphylococcus aureus: Clinical characteristics and antibiotic resistance profile with emphasis on macrolide-lincosamide-streptogramin B resistance.

Healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen worldwide and its multidrug resistance is a major concern. This study aimed to determine the clinical characteristics and antibiotic susceptibility profile of healthcare-associated MRSA with emphasis on resistance to macrolide-lincosamide-streptogramin B (MLSB) phenotypes and vancomycin. This cross-sectional study was carried out between February 2014 and February 2015 across four tertiary care hospitals in Mangalore, South India. Healthcare-associated infections among 291 inpatients at these hospitals were identified according to the Centers for Disease Control and Prevention guidelines. Clinical specimens were collected based on infection type. S. aureus and MRSA isolates were identified and antibiotic susceptibility tests performed using the Kirby-Bauer disk diffusion method. The minimum inhibitory concentration of vancomycin was determined using the Agar dilution method and inducible clindamycin resistance was detected with a double-disk diffusion test (D-test). Out of 291 healthcare-associated S. aureus cases, 88 were MRSA (30.2%). Of these, 54.6% were skin and soft tissue infections. All of the isolates were susceptible to teicoplanin and linezolid. Four MRSA isolates exhibited intermediate resistance to vancomycin (4.6%). Of the MRSA strains, 10 (11.4%) were constitutive MLSB phenotypes, 31 (35.2%) were inducible MLSB phenotypes and 14 (15.9%) were macrolide-streptogramin B phenotypes. Healthcare-associated MRSA multidrug resistance was alarmingly high. In routine antibiotic susceptibility testing, a D-test should always be performed if an isolate is resistant to erythromycin but susceptible to clindamycin. Determination of the minimum inhibitory concentration of vancomycin is necessary when treating patients with MRSA infections.