Abundance of colistin-resistant Escherichia coli harbouring mcr-1 and extended-spectrum ß-lactamase-producing E. coli co-harbouring blaCTX-M-55 or -65 with blaTEM isolates from chicken meat in Vietnam.

Although the spread of plasmid-mediated antibiotic-resistant bacteria is a public health concern, food contamination with plasmid-mediated antibiotic-resistant Escherichia coli in Vietnam has not been well investigated. This study aimed to describe the prevalence of colistin-resistant, carbapenem-resistant, and endemic blaCTX-M in extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Colistin and carbapenem-resistant ESBL-producing E. coli were isolated from chickens in Vietnam and Japan. Colistin-resistant and AmpC/ESBL-producing E. coli (52% and 93%, respectively) were detected in chickens from Vietnam, in comparison to 52.7%, AmpC/ESBL-producing E. coli found in chicken from Japan. Carbapenem-resistant E. coli has not been isolated in Vietnam and Japan. Genotyping revealed that colistin-resistant E. coli harboured mcr-1, and most of the AmpC/ESBL-related genes were blaCTX-M-55 and blaCTX-M-65 together with blaTEM in Vietnamese chickens and blaCMY-2 in Japanese chickens. Multi-drug resistance analysis showed that ESBL-producing E. coli isolates had greater resistance to quinolones, streptomycin, and chloramphenicol than colistin-resistant E. coli isolates from Vietnam, suggesting the selection of multiple antibiotic resistance genes in ESBL-producing E. coli. In conclusion, colistin-resistant E. coli was detected in approximately half of the chicken samples, the majority of which harboured mcr-1. The high prevalence of ESBL-producing E. coli has remained constant in the last 5 years. The predominant blaCTX-M in ESBL-producing E. coli was blaCTX-M-55 or blaCTX-M-65, with the coexistence of blaTEM in Vietnam. These results can be implemented in monitoring systems to overcome the development of antimicrobial resistance.

Multidrug-resistant bacteria with ESBL genes: a growing threat among people living with HIV/AIDS in Nepal.

BACKGROUND: Bacterial opportunistic infections are common in people living with HIV/AIDS (PLHA). Besides HIV-TB co-infection, lower respiratory tract infections (LRTIs) due to multidrug-resistant (MDR) bacteria cause significant morbidity and mortality among PLHA. This study identified bacterial co-infection of the lower respiratory tract and detected plasmid-mediated blaTEM and blaCTX-M genes among Extended-Spectrum ß-Lactamase (ESBL) producing isolates from sputum samples in PLHA. METHODS: A total of 263 PLHA with LRTIs were enrolled in this study, out of which, 50 were smokers, 70 had previous pulmonary tuberculosis, and 21 had CD4 count < 200 cells/µl. Sputum samples collected from PLHA were processed with standard microbiological methods to identify the possible bacterial pathogens. The identified bacterial isolates were assessed for antibiotic susceptibility pattern using modified Kirby Bauer disk diffusion method following Clinical Laboratory Standard Institute (CLSI) guidelines. In addition, plasmid DNA was extracted from MDR and ESBL producers for screening of ESBL genes; blaCTX-M and blaTEM by conventional PCR method using specific primers. RESULTS: Of 263 sputum samples, 67 (25.48%) showed bacterial growth. Among different bacterial pathogens, Klebsiella pneumoniae, (17; 25.37%) was the most predominant, followed by Haemophillus influenzae, (14; 20.90%) and Escherichia coli, (12; 17.91%). A higher infection rate (4/8; 50%) was observed among people aged 61-70 years, whereas no infection was observed below 20 years. About 30.0% (15/50) of smokers, 32.86% (23/70) cases with previous pulmonary tuberculosis, and 52.38% (11/21) with CD4 count < 200 cells/µl had bacterial LRTIs. Among 53 bacterial isolates excluding H. influenzae, 28 isolates were MDR and 23 were ESBL producers. All ESBL producers were sensitive to colistin and polymyxin B. Among ESBL producers, 47.83% (11/23) possessed blaCTX-M, 8.6% (2/23) were positive for blaTEM gene, and 43.48% (10/23) possessed both ESBL genes. CONCLUSION: The increasing rate of MDR bacterial infections, mainly ESBL producers of LRTIs causes difficulty in disease management, leading to high morbidity and mortality of PLHA. Hence, it is crucial to know the antibiogram pattern of the isolates to recommend effective antimicrobial therapy to treat LRTIs in PLHA.

Identification of oral anaerobic bacteria and the beta-lactamase resistance genes from Iranian patients with periodontitis.

OBJECTIVES: The dysbiosis of bacteria and horizontal transfer of antibiotic resistance genes (ARGs) could be highly problematic particularly in the oral environment. Here, we aimed to identify the anaerobic species from patients with periodontitis and to screen the isolates for the ß-lactamase resistance genes, blaTEM, cfxA, its variants, and mobA. METHODS: The 129 samples from periodontal pockets were subjected to anaerobic culture, followed by 16S rRNA gene sequencing, PCR assays for the cfxA, blaTEM, and mobA. The minimum inhibitory concentration (MIC) of amoxicillin, ampicillin, amoxicillin/clavulanate, ampicillin/sulbactam, and cefixime was determined against CfxA producing isolates using MIC Test Strips. RESULTS: The species with frequency higher than 10% were Lactobacillus spp. (26.3%), Streptococcus spp. (18.8%), Leptotrichia wadei (14%) and Veillonella spp. (11.4%). The blaTEM was not found in any of the isolates whereas cfxA was found in 12.5% of isolates including V. parvula, V. rogosae, Prevotella nigrescens and Campylobacter concisus. Of CfxA variants, CfxA2 (90%) was the most frequent one. Among the CfxA producing isolates, the resistance to ampicillin and amoxicillin was observed only in two isolates of P. nigrescens and V. rogosae. CONCLUSIONS: This study showed that various anaerobes species may be involved in the development of periodontitis. Of them, Prevotella and Veillonella species were found to commonly carry cfxA even though they are susceptible to beta-lactams and its combination.

Abundance of extended-spectrum ß-lactamase genes among intestinal Escherichia coli strains from drug users.

Drug users may represent a hidden reservoir of antibiotic resistance genes among their intestinal flora due to the poor hygiene and inappropriate use of antibiotics. Therefore, this study was focused to examine the prevalence of extended-spectrum ß-lactamase (ESBL) genes among intestinal Escherichia coli isolated from drug users in Ahvaz, Iran. Among clients of toxicology laboratory who were confirmed their addiction to each of Morphine, Amphetamine or Methamphetamine, 109 drug users were examined voluntarily for infection with hepatitis B or C using commercial enzyme linked immunosorbent assays (ELISA) method. Their stool specimens were obtained to isolate intestinal E. coli. The disc diffusion and combination disk methods were conducted to demonstrate antibiotic resistance pattern and phenotypically ESBL producers. ESBL-encoding genes (bla-TEM, bla-CTX-M, and bla-SHV) were also examined by PCR. Based on results, hepatitis C infection was more prevalent than hepatitis B among drug users. Of 109 isolates, a total of 57 (52.29%) ESBL positive E. coli were obtained from drug users and bla-TEM gene (60.55%) was found to be the most prevalent type, followed by bla-CTX-M (40.36%) and bla-SHV (39.44%). All isolates represented different resistance levels to tested antibiotics and 54.43% of the ESBL­producing isolates showed multidrug resistance (MDR) and the most frequent MDR pattern was simultaneous resistance to the seven (27.90%) of antimicrobials particularly erythromycin, penicillin, amoxycilin, cefteriaxon, cefotaxim, tetracycline and trimethoprim-Sulfamethoxazole. Fecal carriage of ESBL-production and MDR commensal isolates such as E. coli among drug users underlines the risk of transferring resistance genes between nonpathogenic and pathogenic bacteria.

Genetic characterization of coliform bacterial isolates from environmental water in Thailand.

INTRODUCTION: In contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. METHODS: Coliform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. RESULTS: In 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with ß-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms' wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. CONCLUSIONS: Results obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

Prevalence and distribution of blaCTX-M, blaSHV, blaTEM genes in extended- spectrum ß- lactamase- producing E. coli isolates from broiler farms in the Philippines.

BACKGROUND: Antimicrobial resistance is a worldwide problem causing serious health threats. Escherichia coli is one of the most important bacteria that causes resistance problem. These bacteria produce an enzyme called extended-spectrum ß-lactamase (ESBL) that allows it to become resistant to a wide variety of penicillins and cephalosporins. Currently, no information or published studies on ESBL-producing E.coli in broilers are available in the Philippines. This cross-sectional study was conducted to determine the prevalence and distribution of extended-spectrum ß-lactamase (ESBL)-encoding genes, blaCTX-M, blaSHV, and blaTEM, among E. coli isolates from broiler farms in Luzon, Philippines. RESULTS: Results showed a farm prevalence of 66. 67%. A total of 69 (44.23%) ESBL-producing E. coli were isolated from boot swabs and cloacal swab samples from broiler farms. All major blaCTX-M groups except blaCTX-M-25 group were identified in the isolates. The most prevalent group was blaCTX-M-1, 72.46% (CI: 60.38-82.54%), followed by blaCTX-M-2, blaCTX-M-9 group and blaCTX-M-8. The blaTEM and blaSHV genes were identified in 57.97 and 27.54% of isolates, respectively. The blaCTX-M and blaTEM were the most common gene combinations (33.33%). Coexistence of blaCTX-M types was observed in 50 (73.53%) isolates. CONCLUSION: This study shows the high prevalence, diversity of patterns and coexistence of ESBL genes in the E. coli isolates from cloacal and boot swabs from broiler farms which pose risks of possible transmission to the environment, other animals and human.

Molecular characterization of extended spectrum ß-lactamase-producing Enterobacteriaceae strains isolated from a tertiary care hospital.

Infectious diseases caused by ESBL producing Enterobacteriaceae are an emerging problem worldwide which increases the empirical treatment failure, hospital cost, rate of morbidity and mortality. This also leads to the Hospital infection outbreak. Present study was undertaken to determine the frequency of blaTEM, blaCTX-M and blaSHV genes among Enterobacteriaceae. A total of 751 non-repeated clinical isolates of Enterobacteriaceae family were included in this study. Antibiotic susceptibility test was done and minimal inhibitory concentration (MIC) against four antibiotics was carried out. Five hundred fifteen multi drug resistant isolates were tested for ESBL by CLSI confirmatory method. Isolates showing ESBL positive by phenotypic method were screened for blaTEM, blaCTX-M and blaSHV genes by monoplex PCR. Two blaTEM and two blaCTX-M amplified products were selected randomly for sequencing. Sequencing data was submitted to NCBI data base. Of the 515 MDR isolates, 140 showed ESBL production by phenotypic method. All the ESBL producing isolates showed resistant to ceftazidime (100%). IMP, TGC and CL drugs could be preferred for the treatment of ESBL producers as these drugs showed a lower rate of resistance. blaTEM gene was the predominant (96.42%) followed by blaCTX-M (75%) and blaSHV (17.85%). All the three bla genes were occurred in 22 (17.14%) isolates. All the phenotypically confirmed ESBL producers were found contain any one of the three bla genes. It is concluded from the study that the blaTEM was predominantly found in Enterobacteriaceae and blaCTX-M gene also seemed to emerging.

Evaluation of antibiotic resistant lactose fermentative opportunistic pathogenic Enterobacteriaceae bacteria and blaTEM-2 gene in cephalosporin wastewater and its discharge receiving river.

This study investigated the concentration of cephalosporin, the resistant levels of lactose fermentative opportunistic pathogenic Enterobacteriaceae bacteria (LFOPEB) against seven antibiotics and one cephalosporin-resistant gene in cephalosporin wastewater (CPWW) treatment plant and its discharge receiving river. Although large numbers of bacteria have been removed during the CPWW treatment process, the antibiotic resistant rates of the isolates to ß-lactam antibiotics significantly increased (p = 0.032) after treatment, while the percentage of resistant LFOPEB to non-ß-lactam antibiotics did not change dramatically. Furthermore, the discharge of the effluent of CPWW treatment plant (CPWWeff) led to an obvious increase in the percentages of ß-lactam antibiotic-resistant LFOPEB and relative abundance of the blaTEM-2 gene in the downstream receiving river (RWdown) in comparison with those in the upstream receiving river (RWup). The antibiotic resistant phenotypes of isolates in the influent of CPWW treatment plant (CPWWin), CPWWeff and RWdown appeared to be seriously affected by the cephalosporin residues, which suggested that main antibiotic resistance phenotypes in antibiotic contaminated water were closely associated with its antibiotic composition. Therefore, CPWW treatment process has been proved to result in selective growth of ARB and proliferation of ARG. Besides, CPWWeff was also proved to be an important supplier of ARB and ARG to the receiving river.

Genomic characteristics of two strains of ESBL-producing Klebsiella pneumoniae ST268 isolated from different samples of one patient.

OBJECTIVES: This study reports the whole-genome sequences of two strains of extended-spectrum beta-lactamase (ESBL)-producing and multidrug-resistant (MDR) K. pneumoniae ST268 and explores their acquired antibiotic resistance genes (ARGs) and the mobile genetic elements (MGEs). METHODS: Two strains of K. pneumoniae ST268 were isolated from different samples of one patient. Assessment of antimicrobial susceptibility was performed, and then whole-genome sequencing was conducted. Acquired ARGs, insertion sequences, and transposons harboured by the two strains of K. pneumoniae ST268 were identified, and then the genetic contexts associated with the ARGs were analysed systematically. RESULTS: Two strains of K. pneumoniae ST268 were found to carry the 118.6-kb hybrid IncFIIK:IncQ1:repBR1701 plasmid. All the acquired ARGs carried by the IncF plasmid were found to be situated on the 25.3-kb MDR region bracketed by ISKpn19 and IS26, which was widely present in the plasmids in 14 STs of strains in K. pneumoniae but also in IncF plasmids from Shigella flexneri and Klebsiella quasipneumoniae. Notably, the IncF plasmids harbouring the 25.3-kb MDR region were geographically distributed mainly in China, and the pKP161637-1/pKP160802-1 in our study was the first report on the IncF plasmid carrying the 25.3-kb MDR region bracketed in K. pneumoniae ST268. CONCLUSIONS: Two strains of ESBL-producing K. pneumoniae ST268 with a MDR IncF plasmid were identified in a hospital in China. The ARGs were identified on the 25.3-kb MDR region, bracketed by ISKpn19 and IS26, of the IncF plasmids, which were present not only in the K. pneumoniae but also in the S. flexneri and K. quasipneumoniae.

Occurrence and characteristics of extended-spectrum-ß-lactamase producing Escherichia coli (blaTEM-128) isolated from Mus musculus captured from a veterinary clinic and houses in Tunis, Tunisia.

Antimicrobial resistance in Enterobacteriaceae is a public health problem. Rodents, can be a potential vector for transmission of multidrug resistant bacteria between animals, humans, and environment. The aim of our study was to assess the level of Enterobacteriaceae present in the intestines of rats collected from different locations in Tunisia, then to determine their antimicrobial susceptibility profiles, to screen extended spectrum ß-lactamases-producing strains and determine the molecular mechanism of ß-lactams resistance. Between July 2017 and June 2018, 55 strains of Enterobacteriaceae were isolated from 71 rats captured in various locations in Tunisia. Antibiotic susceptibility testing was performed using the disc diffusion method. Genes encoding ESBL and mcr genes were investigated by RT-PCR, standard PCR and sequencing when these genes were found. Fifty-five strains of Enterobacteriaceae were identified. The overall prevalence of ESBL production found in our study was 12.7 % (7/55) of which two E. coli strains were DDST positive, one isolated from a house-caught rat and one from the veterinary clinic and harbored the blaTEM-128 gene. In addition, the other five strains were DDST negative and harbored the blaTEM gene, including three strains isolated from collective restaurant (n = 2: blaTEM-163; n = 1: blaTEM-1), one strain isolated from the veterinary clinic (blaTEM-82), and one strain isolated from a house (blaTEM-128). The results of our study suggest that rodents may play a role in the spread of antimicrobial resistant E. coli, highlighting the need to protect the environment and monitor antimicrobial resistant bacteria in rodents to prevent their spread to other wildlife and humans.

Microbiological quality, antibiotic resistant bacteria and relevant resistance genes in ready-to-eat Pacific oysters (Magallana gigas).

Oysters are a highly valued seafood but can endanger public health, if they are eaten raw or barely cooked. We evaluated the microbiological quality of Pacific oysters (Magallana gigas) by international standard methods in four groups (each with four to five animals) acquired from supermarkets and directly from a farm producer. Most of the groups presented satisfactory microbiological quality. In two groups of oysters, 'questionable' or 'unsatisfactory' quality was observed for the coagulase-positive Staphylococcus parameter. Culture-based methods did not detect Salmonella spp. or enteropathogenic Vibrio spp., but Vibrio alginolyticus, a potential foodborne pathogen, was identified by molecular analysis. Fifty strains, belonging to 19 species, were isolated in antibiotic-supplemented media, and their antibiotic susceptibility profile was evaluated. Genes coding for ß-lactamases were searched by PCR in bacteria showing resistance phenotype. Decreased susceptibility or resistance to distinct antibiotics were observed for bacteria from depurated and non-depurated oysters. The blaTEM gene was identified in Escherichia fergusonii and Shigella dysenteriae strains, which showed multidrug-resistant phenotypes. The possibility that oysters might be a source of antibiotic-resistant bacteria/antibiotic resistance genes is of great concern and highlights the need for stricter controls and preventative measures to mitigate and counteract the dissemination of antibiotic resistance across the food chain.

Molecular Characterization of ESBLs and QnrS Producers From Selected Enterobacteriaceae Strains Isolated From Commercial Poultry Production Systems in Kiambu County, Kenya.

Background: The emergence and spread of Extended-spectrum ß-lactamases (ESBLs) in Enterobacteriaceae through the plasmid-mediated exchange have become a major threat to public health by complicating the treatment of severe infections in both animals and humans. Therefore, the current study focused on evaluating the manifestation of ESBLs production from the fecal isolates of E. coli, Shigella spp, Salmonella spp, and Klebsiella spps in commercial poultry production systems of Kiambu County, Kenya. Materials and methods: Out of 591 isolates identified as E. coli, Shigella spp, Salmonella spp, and Klebsiella spps from 437 fecal samples, only 78 were phenotypically suggestive to be ESBL producers. The possible ESBL producers were screened for the presence of blaTEM, blaCTX-M, blaOXA, and blaSHV using the PCR technique. These isolates were also screened for carriage of the QnrS gene that confers resistance to the fluoroquinolone class of drugs. Results: The most detected ESBL gene from the isolates was blaOXA (n = 20; 26%), followed by blaTEM (n = 16, 21%), with the majority of them detected in E. coli. The blaCTX-M was identified in all the 4 enteric's bacteria-type isolates tested. Three E. coli and Salmonella spp respectively were found to harbor all the 5 antimicrobial resistance (AMR) gene types. The blaTEM, blaOXA, blaSHV, and QnrS genes were not detected from Klebsiella and Shigella spps. Additionally, most of the AMR gene co-carriage was detected in both E. coli and Salmonella spps as follows blaTEM + blaOXA (n = 4); blaTEM + QnrS (n = 3); blaTEM + blaOXA + QnrS (n = 3), concurrently. Conclusion: Our findings highlight the significance of commercial poultry production in disseminating transferable antibiotic resistance genes that act as potential sources of extensive drug resistance in livestock, humans, and the environment, leaving limited therapeutic options in infection management.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996-2018.

OBJECTIVES: The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. METHODS: Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. RESULTS: Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 ß-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. CONCLUSION: The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum ß-lactamase (ESBL).

Molecular characterization of decreased susceptibility to ceftriaxone and genotyping of Neisseria gonorrheae isolates in New Delhi, India.

Data on genetic characteristics of Neisseria gonorrhoeae isolates exhibiting decreased susceptibility to extended-spectrum cephalosporins in India is deficient. In this study, we have sequenced penA, porB, mtrR and ponA and blaTEM genes in 70 clinical isolates of NG with varying ceftriaxone MICs. Amongst these, 22 (31.4%) were PPNG. Additionally, N. gonorrheae Multiantigen Sequence Typing was performed. Fisher exact and χ2 were used to evaluate significance of mutations with MICs. A total of six non-mosaic penA (Penicillin binding protein 2 [PBP2]) amino acid patterns were seen (II, IV, IX, XII, XIX, XXII) of which, pattern IX was significantly associated with decreased susceptibility to ceftriaxone. Other significant associations were noted in porB & mtrR genes. There were no mutations in blaTEM gene. ST6069 was significantly associated with decreased susceptibility to ceftriaxone. To conclude, development of decreased susceptibility to ceftriaxone in gonococci involves cumulation of different mutations in the four chromosomal genes investigated.

Genomic characterisation and epidemiology of nosocomial Serratia marcescens isolates resistant to ceftazidime and their plasmids mediating rare blaTEM-61.

OBJECTIVES: We determined the whole DNA sequences of plasmids carrying a rare extended-spectrum ß-lactamase gene (blaTEM-61) to precisely understand the spread of resistance among nosocomial Serratia marcescens populations. METHODS: Twenty non-duplicate ceftazidime-resistant S. marcescens nosocomial isolates (ceftazidime MICs, 32 to >128 mg/L) collected over 1 year were pulsotyped and nucleotide sequences of the blaTEM-61 gene and its promoter region were determined. Twelve representative isolates were analysed by whole-genome sequencing. RESULTS: The 20 isolates comprised two distinct pulsotypes: I (14 isolates) and II (6 isolates). They all contained the blaTEM-61 gene. A polymorphism in the repeat number of a 15-nucleotide sequence (5'-ATGTCATGATAATAA-3') was found in the promoter region of blaTEM-61; two, three and four repeat units were found in 6, 12 and 2 isolates, respectively. Single nucleotide polymorphism (SNP)-based phylogenetic analysis of 12 isolates revealed that 7 isolates of pulsotype I (12-44 SNP differences) and 5 isolates of pulsotype II (15-55 SNP differences) formed two distinct clusters of genotypes 1 and 2, respectively. All 12 isolates harboured a plasmid carrying the Tn1-blaTEM-61 element, although they were slightly different in size (78 883 bp, 78 898 bp and 78 913 bp) owing to differences in the number of 15-bp repetitive sequences. A 42 542-bp broad-host-range plasmid carrying the Tn1-blaTEM-61 element was also found in one of the isolates. CONCLUSIONS: We characterised a plasmid-encoded novel Tn1-blaTEM-61 element and transposon-dependent mechanisms underlying the propagation of antibiotic resistance, together with repeated new polymorphic 15-bp units in the promoter of blaTEM-61.

Isolation and antimicrobial resistance of motile Salmonella enterica from the poultry hatchery environment.

Salmonella is a globally distributed major food-borne pathogen and poultry is one of the predominant sources of salmonellosis in humans. To investigate the presence of motile Salmonella in the poultry hatchery environment, we collected 97 fluff samples from four selected broiler breeder chicken hatcheries from Chattogram, Bangladesh during July-December 2015. To isolate motile Salmonella enterica, we used conventional bacteriological techniques followed by serological verification using anti-Salmonella Poly A-E serum and species confirmation by conventional PCR assay. Antimicrobial susceptibility testing by Kirby-Bauer disc diffusion method for 10 commonly used antibiotics was performed on all isolates. Isolates displaying phenotypic resistance to ampicillin were tested by PCR for blaTEM gene, whereas those resistant to tetracycline were tested for the presence of tetA, tetB and tetC genes. A total of 24 samples (24.7%; 95% CI: 16.5-34.5, N = 97) from 3 hatcheries were positive for motile Salmonella. Of them, 21 (87.5%) and 12 (50.0%) were resistant to ampicillin and tetracycline, respectively, 9 (37.5%) to nalidixic acid and sulphamethoxazole/trimethoprim. No resistance was detected to ceftriaxone, cefoxitin, gentamicin, neomycin, ciprofloxacin and colistin. Ten (42%) of 24 isolates from 2 hatcheries were multi-drug resistant (i.e. resistant to ≥ 3 antimicrobial classes). Six of 21 ampicillin resistant isolates contained blaTEM gene and 10 of 12 tetracycline resistant isolates contained tetA gene. This study highlights the circulation of multi-drug resistant motile Salmonella in the hatchery environment for the first time in Bangladesh. Further epidemiological and molecular studies are therefore needed to identify the serotypes and source of the bacteria in hatcheries.

Lower respiratory tract isolates of non-typeable Haemophilus influenzae in Western Sichuan, China: Antimicrobial susceptibility, mechanisms of ß-lactam resistance and decade changes.

OBJECTIVES: The aims of this study were to analyse the serotypes of epidemic Haemophilus influenzae and changes in mechanisms of ß-lactam resistance over the past decade. RESULTS: Haemophilus influenzae isolates in Western Sichuan from 2013-2014 were non-typeable H. influenzae (NTHi). ß-Lactam MICs for NTHi isolated during 2013-2014 were significantly higher than those from 2003-2004 (P < 0.05). Of 274 NTHi, 141 (51.5%) were ß-lactamase-positive (TEM-1 type). There were 35 amino acid (AA) substitutions in ftsI among NTHi isolated from 2013-2014. However, NTHi isolates from 2003-2004 had only nine AA substitutions. Ordered multiple classification logistic regression analysis showed that different AA substitution patterns in ftsI had different effects on ß-lactam MICs. The main factors affecting the ampicillin MIC were the mutations R517H (OR = 6.999), L389F (OR = 7.128), N526K (OR = 4.660) and D350N (OR = 0.450). The main factor influencing the amoxicillin/clavulanic acid MIC was an N526K mutation (OR = 9.349). The main factors affecting the cefuroxime MIC were the mutations S357N (OR = 37.453) and N526K (OR = 14.816). Compared with 2003-2004, gBLNAR and gBLPAR isolated from 2013-2014 increased significantly from 13.0% (7/54) and 9.3% (5/54) to 38.2% (84/220) and 45.5% (100/220), respectively (P < 0.001). In the 'others' group of ftsI gene mutations, 13 NTHi had the same ftsI gene mutation pattern and 24 AA substitutions. CONCLUSION: These results confirm that ß-lactam-resistant NTHi isolates increased rapidly. AA substitutions in ftsI were more complex and diversified in 2013-2014.

IncFII plasmid carrying antimicrobial resistance genes in Shigella flexneri: Vehicle for dissemination.

OBJECTIVES: Plasmids harbouring antimicrobial resistance determinants in clinical strains are a significant public-health concern worldwide. The present study investigated such plasmids in clinical isolates of Shigella flexneri. METHODS: A total of 162 Shigella isolates were obtained from stool specimens in the year 2015. Among the 70 multidrug-resistant (MDR) Shigella spp., 27 S. flexneri isolates were randomly selected for further characterisation. Antimicrobial resistance genes (ARGs) and plasmid incompatibility (Inc) types were analysed. RESULTS: IncFII plasmids were found in 63% (17/27) of the studied S. flexneri isolates. ARGs such as dhfr1a (81%), sulII (74%), blaOXA (74%), blaTEM (33%), blaAmpC (30%), qnrS (15%) and qnrB (4%) were identified by PCR, whereas blaCTX-M was not detected. Next-generation sequencing of a representative S. flexneri IncFII-type plasmid (pSF470) revealed the presence of blaTEM1-B, blaDHA-1, qnrB10, mphA, sulI, sulII, strA, strB and tetR ARGs along with the intI1 integrase gene. In addition, pMLST analysis showed that the replicon belonged to F2:A-:B- type. CONCLUSIONS: This study helps to know the prevalent plasmid types in MDR Shigella isolates and will improve our understanding of resistance dissemination among enteric bacteria. ARGs in plasmids further highlight the importance of such studies in enteric bacteria.

Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the blaTEM gene.

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Detection and genetic characterization of extended-spectrum beta-lactamases producers in a tertiary care hospital.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing organisms inactivate extended beta-lactam antibiotics and monobactams and also exhibit coresistance to many other classes of antibiotics. The present study was carried out to assess the prevalence of the ESBLs and to determine the most prevalent genotype in our hospital. MATERIALS AND METHODS: All clinically significant Gram-negative isolates were identified, and their antimicrobial susceptibility testing was done by Kirby-Bauers' disc diffusion method. ESBL detection was confirmed by minimal inhibitory concentration method using agar dilution technique for those who screened positive by ceftazidime (30 µg) disc. Further, the established ESBL-positive isolates were subjected to genotyping for bla TEM, bla CTX-M, and bla SHV genes by using conventional polymerase chain reaction. RESULTS: Escherichia coli was the most common (28.84%) Gram-negative bacillus followed by Klebsiella pneumoniae (18.07%), while Pseudomonas spp. (9.61%) was the most commonly identified nonfermenter. ESBL production was detected in 160 (30.8%) isolates. Klebsiella oxytoca (46.7%) followed by E. coli (44%) were the common ESBL producers. Most predominant ESBL gene was bla TEM, found in 122 (76.25%) isolates. Combinations of two genes were seen in 109 (68.1%) isolates, the most common (43.12%) combination being blaTEM and blaCTX-M. In this study, 16 (10%) strains had all the three types of genes. Most of the isolated Gram-negative bacilli (GNB) were sensitive to amikacin, imipenem, and colistin. CONCLUSION: In our study, the 30.8% of GNB were ESBL producers. This is the only study that shows that TEM is the most prevalent ESBL genotypes in our area. Of concern is a good number of isolates showing all three patterns of genes (TEM, SHV, and CTX-M). Amikacin, imipenem, and colistin were the most useful antibiotics in our setup.