Outbreak of Multidrug-resistant Acinetobacter baumannii in a Tertiary Health Center from Northwestern Nigeria.

Background: In spite of its global notoriety and WHO alarm, Acinetobacter baumannii is still an understudied critical-priority pathobiont in Nigeria. We characterized its antimicrobial susceptibility profile and resistance genes during an outbreak. Materials and Methods: This cross-sectional study involved collection of patients' urine samples and swabs from unit staff's hands and ward environments for the identification of A. baumannii strains using standard morphologic and biochemical methods. The disk diffusion method was used to assess the antimicrobial susceptibility profile of the isolates with the production of extended-spectrum beta-lactamases (ESBLs) confirmed by the combined disk test screening method. Characterization of the resistance genes of the ESBL producers was carried out using polymerase chain reaction polymerase chain reaction technique. Results: A.total of eight (six clinical and two nonclinical) A. baumannii isolates were identified. The overall isolate susceptibility and resistance rates to all the antimicrobial agents was 56.3% (27/48) and 35.4% (17/48), respectively. Similarly, all (8/8; 100.00%) isolates were susceptible to meropenem and 75.0% (6/8) to ampicillin-sulbactam while 62.5% (5/8) were resistant to trimethoprim-sulfamethoxazole and 50.0% (4/8) to each of ciprofloxacin and ceftazidime. In addition, 37.5% (3/8) of the isolates were multidrug resistant (MDR) with nonclinical isolates exhibiting more antimicrobial resistance than their clinical counterparts (9/12%-75.0% vs. 8/36%-22.2%). Phenotypic detection and molecular characterization revealed three ESBL-producing isolates that each harbored blaSHV and blaTEM genes with blaCTX-M gene being absent. Conclusion: MDR strains of A. baumannii harboring blaSHV and blaTEM genes were recovered from clinical and environmental sources during the outbreak, which was contained with preventive measures recommended.

Résumé En dépit des alertes faites par l'organisation mondiale de la Santé (OMS), Acinetobacter baumannii demeure un pathobiont sous-étudié et très peu priorisé au Nigeria. Nous avons procedé à sa caractérisation phénotypique et génotypique en dressant son profil de sensibilité aux antimicrobiens et ainsi que les gènes de résistance impliqués au cours d'une épidémie. Matériel et méthodes: Cette étude transversale a consisté à collecter des échantillons d'urine de patients et des écouvillons des mains du personnel des soins et de l'environnement hospitalier. L'identification des souches d' A. baumannii était faite par des méthodes bactériologiques standard. le profil de sensibilité aux antimicrobiens des isolats a été faite par la méthode de diffusion de disque , les bêta-lactamases à spectre étendu (BLSE) étaient recherchée par la méthode de dépistage combinée de disque ainsi que leur caractérisation moléculaire par la mise en évidence des gènes de résistance BLSE à l'aide d'une PCR (réaction en chaîne par polymérase). Résultats: Au total, huit isolats d'A. baumannii (6 cliniques et 2 de l'environnement) ont été identifiés. Les taux globaux de sensibilité et de résistance des isolats à tous les agents antimicrobiens étaient respectivement de 56,3 % (27/48) et de 35,4 % (17/48). De même, tous les isolats (8/8 ; 100,00 %) étaient sensibles au méropénème et 75,0 % (6/8) à l'ampicilline-sulbactam, tandis que 62,5 % (5/8) étaient résistants au triméthoprime-sulfaméthoxazole et 50,0 % (4/8) à la ciprofloxacine et à la ceftazidime. En outre, 37,5 % (3/8) des isolats étaient multirésistants (MDR), les isolats non cliniques présentant une plus grande résistance aux antimicrobiens que leurs homologues cliniques (9/12 %-75,0 % contre 8/36 %-22,2 %). La détection phénotypique et la caractérisation moléculaire ont révélé trois isolats producteurs de BLSE qui hébergeaient chacun les gènes blaSHV et blaTEM, le gène blaCTX-M étant absent. Conclusion: Des souches multirésistantes d'A. baumannii portant les gènes blaSHV et blaTEM ont été identifiées sur des prélevements cliniques et environnementaux au cours de l'épidémie, qui a été gerée grâce aux mesures préventives recommandées. Mots-clés: Surveillance de la résistance aux antimicrobiens, blaSHV carbapénème, pathogène ESKAPE, infections associées aux soins de santé, pratiques de prévention et de contrôle des infections, one health, uropathogènes.

Dynamic within-host cefiderocol heteroresistance caused by blaSHV-12 amplification in pandrug-resistant and hypervirulent Klebsiella pneumoniae sequence type 11.

AIMS: Although cefiderocol (FDC) is not prescribed in China, FDC-resistant pandrug-resistant hypervirulent Klebsiella pneumoniae (PDR-hvKp) is emerging. In this study, we performed FDC susceptibility testing of clinical Kp isolates to explore the prevalence of FDC-resistant isolates and the mechanism of FDC-resistance. METHODS: We retrospectively selected 151 carbapenem-resistant Kp isolates to assess FDC susceptibility. Seven isolates harboring blaSHV-12 from two patients were enrolled for whole-genome sequencing. The antimicrobial resistance, virulence, blaSHV-12 expression, and fitness costs in different media were examined. The amplification of blaSHV-12 was further investigated by qPCR and long-read sequencing. RESULTS: The 151 isolates showed a low MIC50/MIC90 (1/4 mg/L) of FDC. The seven isolates were ST11 PDR-hvKp, and two represented FDC-resistance (MIC=32 mg/L). The IncR/IncFII plasmids of two FDC-resistant isolates harbored 6 and 15 copies of blaSHV-12, whereas four FDC-susceptible isolates carried one copy and one harbored three copies. These blaSHV-12 genes concatenated together and were located within the same 7.3 kb fragment flanked by IS26, which contributed to the increased expression and FDC resistance without fitness costs. The amplification of blaSHV-12 and FDC resistance could be induced by FDC in vitro and reversed during continuous passage. CONCLUSIONS: The amplification of blaSHV-12 and the consequent dynamic within-host heteroresistance are important concerns for the rational application of antibiotics. Long-read sequencing might be a superior way to detect resistance gene amplification rapidly and accurately.

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.

Detection of blaTEM, blaCTX-M, blaCMY, and blaSHV Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolated from Migratory Birds Travelling to Bangladesh.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli cause severe health hazards. Migratory birds are reservoirs and transmitters of many pathogens including ESBL-producing E. coli. To examine migratory birds as potential carriers of ESBL-producing E. coli and E. coli-carrying antibiotic resistance genes, 55 PCR-positive E. coli isolates were screened using the disk diffusion method, double-disk synergy test, and further polymerase chain reaction (PCR) tests. Genes encoding resistance to tetracycline [tetA, 100% (35/35); tetB, 31.43% (11/35)], fluoroquinolone [qnrA, 35.71% (10/28); qnrB, 25% (7/28)], and streptomycin [aadA1, 90.24% (37/41)] were detected in the isolated E. coli. Of the 55 E. coli isolates, 21 (38.18%) were ESBL producers, and all of them were multidrug resistant. All the ESBL-producing E. coli isolates harbored at least two or more beta-lactamase genes, of which blaTEM, blaCMY, blaCTX-M, and blaSHV were detected in 95.24%, 90.48%, 85.71%, and 42.86% of isolates, respectively. All the beta-lactamase genes were present in four of the ESBL-producing E. coli isolates. Furthermore, 95.24% of ESBL-producing E. coli isolates were positive for one or more antibiotic resistance genes. To the best of our knowledge, this is the first study to detect E. coli-carrying antibiotic resistance genes including beta-lactamase blaCMY and blaSHV originating from migratory birds in Bangladesh. These results suggest that migratory birds are potential carriers of ESBL-producing E. coli along with other clinically important antibiotic resistance genes which may have detrimental impacts on human health.

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.

A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying blaSHV-12 from horses in Sweden.

OBJECTIVES: The aim of this study was to investigate the occurrence of the newly described transferable colistin resistance gene mcr-9 in extended-spectrum ß-lactamase (ESBL)-producing clinical Enterobacteriaceae isolates from horses in Sweden. METHODS: A total of 56 whole-genome sequenced ESBL-producing Enterobacteriaceae isolates from horses were subjected to in silico detection of antimicrobial resistance genes and identification of plasmid replicons types. The colistin minimum inhibitory concentration (MIC) for mcr-positive isolates was determined by broth microdilution. Relatedness between Enterobacteriaceae carrying mcr genes was determined by multilocus sequence typing (MLST) and core genome MLST. RESULTS: Thirty ESBL-producing Enterobacteriaceae isolates from horses were positive for the colistin resistance gene mcr-9. These isolates included Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca and Citrobacter freundii and belonged to diverse MLST sequence types within each species. Two of the mcr-9-containing isolates originated from the same horse. All mcr-9-positive isolates had colistin MICs below or equal to the EUCAST epidemiological cut-off value of 2 mg/L and were negative for the two potential regulatory genes qseB-like and qseC-like for mcr-9. Except for one isolate carrying only blaTEM-1B, all of the isolates carried blaSHV-12 and blaTEM-1B, and were all considered multidrug-resistant as they harboured genes encoding resistance to aminoglycosides, chloramphenicol, fosfomycin, macrolides, quinolones, sulfonamides, trimethoprim and tetracyclines. Plasmid replicon types IncHI2 and IncHI2A were detected in all mcr-9-positive isolates. CONCLUSION: The occurrence of mcr-9 was common among clinical ESBL-producing Enterobacteriaceae isolates from horses in Sweden and was linked to the ESBL-encoding gene blaSHV-12 and plasmid replicon types IncHI2 and IncHI2A.

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.

Distribution of resistance genes encoding ESBLs in Enterobacteriaceae isolated from biological samples in health centers in Ouagadougou, Burkina Faso.

OBJECTIVE: Resistance to antibiotics most especially third generation cephalosporins has assumed a worrisome dimension globally. Genes conferring these resistance which are mediated by enzymes known as extended spectrum beta-lactamases (ESBLs) are now wide spread among several Enterobacteriaceae species. However there is paucity of data regarding the distribution of these genes in Burkina Faso. Hence this prospective study aims to determine the prevalence and distribution of ESBL encoding genes in ESBL producing Enterobacteriaceae strains isolated from clinical samples of patients attending the three major hospitals in Ouagadougou Burkina Faso. RESULTS: ESBL-encoding genes were assayed in 187 ESBL producing Enterobacteriaceae strains. Among these isolates, the prevalence of ESBL-producing strains with blaTEM, blaSHV and blaCTX-M genes were 26.2% (49/187), 5.9% (11/187) and 40.1% (75/187) respectively. The association of ESBL encoding genes with health centers was statistically significant (p = 0.0209). Approximately 39.6% of E. coli harbored CTX-M and Klebsiella spp. 5.9%. This study demonstrates the dissemination of TEM, SHV and CTX-M genes in ESBL producing Enterobacteriaceae strains in Ouagadougou. Continuous spread of these bacteria poses great public health risk, thus increased surveillance and regulation of antibiotics use is imperative in Burkina Faso.

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.

Extended Spectrum Beta-Lactamase (ESBL)-producing bacteria isolated from hospital wastewaters, rivers and aquaculture sources in Nigeria.

Untreated wastewater is a risk factor for the spread of antibiotic resistance in the environment. However, little is known about the contribution of untreated wastewater to the burden of antibiotic resistance in the Nigerian environment. In this study, a total of 143 ceftazidime-/cefpodoxime-resistant bacteria isolated from untreated wastewater and untreated wastewater-contaminated surface and groundwater in Nigeria were screened for extended-spectrum ß-lactamase (ESBL) genes, integrons and integron gene cassettes by PCR. The genetic environment of bla CTX-M-15 was mapped by PCR and potentially conjugative plasmids were detected among the isolates by degenerate primer MOB typing (DPMT). ESBL production was confirmed in 114 (79.7%) isolates and ESBL genes (bla SHV, bla CTX-M-15 and bla TEM) were detected in 85 (74.6%) ESBL-producing isolates. bla CTX-M-15 was associated with ISEcp1 and with orf477 in 12 isolates and with ISEcp1, IS26 and orf477 in six others. To the best of our knowledge, this is the first report of bla CTX-M-15 in hand-dug wells and borehole serving as sources of drinking water and a first report of the genetic environment of bla CTX-M-15 in environmental bacteria from Nigeria. The results of this study confirm untreated wastewater as an important medium for the spread of ESBL-producing bacteria within the Nigerian environment. Hence, the widespread practice of discharging untreated wastewater into the aquatic ecosystem in Nigeria is a serious risk to public health.

Prevalence of TEM, SHV, and CTX-M genes of extended-spectrum ß-lactamase-producing Escherichia coli strains isolated from urinary tract infections in adults.

Urinary tract infections (UTIs) are one of the major sources of widespread infectious diseases in the community as well as in the hospitals which increase the cause of morbidity and mortality. Prevalence of extended-spectrum-ß-lactamase (ESBL)-producing uropathogenic E. coli isolates has been found to be increased rapidly across the world. The present study was undertaken to find out the frequency of bla TEM, bla CTX-M, and bla SHV genes among E. coli isolates from UTI and detect their sensitivity pattern. A total of 112 non-repeated E. coli isolates obtained from urine samples of UTI diagnosed patients were included in this study. Antibiotic susceptibility test was done by disc diffusion method. Seventy seven (68.75%) isolates were MDR and tested for ESBL. ESBL-positive isolates were screened for bla TEM, bla CTX-M, and bla SHV genes by monoplex PCR (polymerase chain reaction). Among 46 ESBL-producing E. coli isolates, 8.69% harboured all the three bla genes. The bla TEM was the predominant (93.47%) gene followed by bla CTX-M (82.6%) and bla SHV (4.34%). It can be concluded that the prevalence of MDR (multidrug resistance) ESBL-producing E. coli appears to be high and the highest identified gene was bla TEM. The knowledge of resistance pattern can help physician's select suitable empirical antibiotic regimens, so that antibiotics showing high-resistance pattern can be avoided.

A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine.

Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae are difficult to differentiate phenotypically, leading to misinterpretation of their infection prevalence. We propose a multiplex PCR for blaSHV, blaLEN and blaOKP and their flanking gene (deoR). Since this scheme focuses only on chromosomal genes, it will be feasible for Klebsiella identification in the clinical routine.

Identification and molecular characterization of Escherichia coli blaSHV genes in a Chinese teaching hospital.

Escherichia coli (E. coli) commonly reside in human intestine and most E. coli strains are harmless, but some serotypes cause serious food poisoning. This study identified and molecularly characterized blaSHV genes from 490 E. coli strains with multi-drug resistance in a hospital population. PCR and molecular cloning and southern blot were performed to assess functions and localizations of this resistant E. coli gene and the pulsed-field gel electrophoresis (PFGE) was utilized to demonstrate the clonal relatedness of the positive E. coli strains. The data showed that 4 of these 490 E. coli strains (4/499, 0.8%) carried blaSHV genes that included EC D2485 (blaSHV-5), EC D2487 (blaSHV-5), EC D2684 (blaSHV-11) and EC D2616 (blaSHV-195, a novel blaSHV). Analysis of blaSHV open-reading frame showed that blaSHV-5 had a high hydrolysis activity to the broad-spectrum penicillin (ampicillin or piperacillin), ceftazidime, ceftriaxone, cefotaxime and aztreonam. blaSHV-195 and blaSHV-11 had similar resistant characteristics with high hydrolysis activities to ampicillin and piperacillin, but low activities to cephalosporins. Moreover, the two blaSHV-5 genes were located on a transferable plasmid (23kb), whereas the other two blaSHV variants (blaSHV-11 and blaSHV-195) seemed to be located in the chromosomal material. Both EC D2485 and EC D2487 clones isolated in 2010 had the same DNA finger printing profile and they might be the siblings of clonal dissemination. The data from the current study suggest that the novel blaSHV and clonal dissemination may be developed, although blaSHV genes were infrequently identified in this hospital population. The results of the work demonstrate the necessity for molecular surveillance in tracking blaSHV-producing strains in large teaching hospital settings and emphasize the need for epidemiological monitoring.

Current status of extended spectrum ß-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan.

Enterobacteriaceae producing extended spectrum ß-lactamase (ESBL) are distributed worldwide. In this study, 114 ESBL-producing Enterobacteriaceae were isolated by analyzing 1672 clinical isolates of Enterobacteriaceae collected from an Okinawa prefectural hospital in Japan between June 2013 and July 2014. The overall prevalence of ESBL-producing Enterobacteriaceae was 6.8%; the prevalence of different bacterial species among the ESBL-producing isolates was as follows: 11.5% Escherichia coli (90 of 783 isolates), 6.2% Klebsiella pneumoniae (19 of 307 isolates), and 11.1% Proteus mirabilis (5 of 45 isolates). The ESBL types blaCTX-M-1, -3, -15, -2, -14, -27, and mutants of blaSHV-1 were detected. Among them, blaCTX-M-15 (33.3%), blaCTX-M-14 (27.8%) and blaCTX-M-27 (33.3%) were dominant in the E. coli isolates, whereas a blaSHV mutant which possessed four mutations (Tyr7Phe, Leu35Gln, Gly238Ser and Glu240Lys) in the amino acid sequence of SHV-1 dominated in the K. pneumoniae isolates (11 of 19, 57.9%). The pandemic E. coli ST131 clone was found to constitute 3.3% of the overall examined isolates and 62.2% of the ESBL-producing E. coli isolates. Our results suggest that the genetic combination of blaCTX-M, and blaSHV and antibiotics-resistant profile were different from that in other regions such as other areas of Japan, Asia, Europe, and North America, especially in the ESBL-producing K. pneumoniae isolates and in the E. coli B2-O25b-ST131 isolates possessing blaCTX-M-15 (40.7% of the E. coli B2-O25b-ST131 isolates). Taken together, our results indicate that the ESBL-producing Enterobacteriaceae in Okinawa, Japan, might be of a unique nature.

Phenotypic and molecular characterization of nosocomial K. pneumoniae isolates by ribotyping.

PURPOSE: To characterize clinical isolates of Klebsiella pneumoniae by phenotypic and genotypic methods. MATERIAL/METHODS: Over a 12-month period, 52 isolates of K. pneumoniae were isolated. Of these 52 isolates, 7 were isolated over a period of 21 days from a suspected outbreak in the Neonatal Intensive Care Unit (NICU) and 45 from sporadic cases occurring in different wards of hospital were analysed. RESULTS: The prevalence of K. pneumoniae isolates was 4% (52/1295). Quinolones, aztreonam and amikacin showed the greatest efficacy showing >85% sensitivity. Of the 52 isolates of K. pneumoniae, 8 (15.4%) isolates were positive for ESBL-production. Among the ESBL-producing K. pneumoniae, two out of 8 (25%) and 6 out of 8 (75%) were positive for (bla)SHV and (bla)CTX-M genes respectively. Ribotyping identified 30 distinct ribogroups among 52 isolates evaluated. Seven NICU outbreak isolates were divided into 2 ribotypes, as many as 6 belonged to one ribotype while one isolate which was isolated a week later was of a different ribotype, indicating the termination of the outbreak in the NICU. The outbreak in the NICU thus, was shown to have been caused by a single clone. CONCLUSIONS: A high discriminatory power, ease of interpretation coupled with excellent reproducibility and stability make ribotyping a very useful technique for investigating the molecular epidemiology of nosocomial infections caused by K. pneumoniae. A regular surveillance of hospital associated infections including monitoring antibiotic sensitivity pattern of K. pneumoniae, ESBL-production and molecular characterization is mandatory to control the spread of multidrug-resistant and ESBL-producing K. pneumoniae and for epidemiological purposes especially in outbreak situations.

Genetic and structural insights into plasmid-mediated extended-spectrum ß-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients.

Resistance to third-generation cephalosporins (3GCs) mediated by extended-spectrum ß-lactamases (ESBLs) in pathogenic Enterobacteriaceae is considered a major public health threat in India. This study deals with the detection of plasmid-mediated blaCTX-M, blaSHV and blaOXA genes, understanding their contribution to the ESBL phenotype, and their molecular interaction with 3GCs. More than 87% of isolates showed 3GC resistance, with ESBL production in 60.0% of Escherichia coli and 47.7% of Klebsiella pneumoniae. Molecular characterisation revealed the presence of blaCTX-M-15 (29.8%), blaCTX-M-truncated (1.3%), blaCTX-M-27 (0.7%), blaSHV-1 (20.5%), blaSHV-11 (2.0%), blaSHV-42 (0.7%) and blaOXA-1 (9.9%), among which blaCTX-M variants and blaSHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all blaCTX-M variants, blaSHV-11 and blaSHV-42. The instability index and Gibbs free folding energy change (ΔΔG) predicted decreased stability of SHV-11 and SHV-42. Mutations of CTX-M-truncated, SHV-11 and SHV-42 located in the core region of the enzymes were found to be functional/pathogenic in nature. The catalytic pockets of CTX-M-15 and SHV-42 had the greatest molecular surface area, which might explain their expanded substrate spectrum towards oxyimino-cephalosporins. Molecular dynamics analysis indicated different structural flexibility of CTX-M-truncated compared with the other enzymes. Amino acid alterations resulted in a change of orientation of catalytic residues of class A ß-lactamases that might affect their catalytic processes. Molecular interactions revealed higher catalytic efficiency (ΔG and Km) of CTX-M-15, CTX-M-truncated, CTX-M-27, SHV-11 and SHV-42 compared with their respective wild-types. This study provides useful insights into ESBL production of pathogenic Enterobacteriaceae in India that might help in the development of new antibiotics.

Diversity of ß-lactamases produced by imipenem resistant, Pseudomonas aeruginosa isolates from the bloodstream.

INTRODUCTION: The emergence of imipenem non-susceptible Pseudomonas aeruginosa isolates is a matter of great concern because these isolates can become resistant to all available antibiotics. This study conducted to characterize ß-lactamase genes in imipenem resistant P. aeruginosa isolates from bloodstream. METHODS: 56 non-duplicate clinical isolates of P. aeruginosa were collected in Tehran hospitals. Antibacterial susceptibility was determined by disk diffusion and MIC methods. ESBL and MBL production was confirmed by combined disk. ß-Lactamase classes A, B and D genes were identified by PCR. RESULTS: Seventeen (30.3%) isolates were imipenem resistant for which 16 isolates simultaneously were resistant to all tested antibiotics. While among 39 imipenem susceptible isolates, only two isolates were resistant to all tested antibiotics. In imipenem resistant isolates, blaTEM, blaSHV and blaOXA-10 were found in 41.1% of isolates and blaVIM, blaIMP and blaPER were identified in 47%, 11.7% and 5.8% of isolates respectively, while in imipenem susceptible isolates, blaTEM, blaSHV and blaOXA-10 were determined in 2.5%, 7.6% and 33.3% of isolates, respectively. The imipenem resistant isolates had been recovered mostly (67.7%) from patients in the Burn hospital. CONCLUSION: The result of this study indicated the emergence of multidrug resistant MBL and non-MBL producing P. aeruginosa, particularly in the Burn hospital and blaVIM was dominant ß-lactamase genes in imipenem resistant isolates. The isolation of carrier patients may lead to prevent a further dissemination.

Microbiological quality of ready-to-eat salads: an underestimated vehicle of bacteria and clinically relevant antibiotic resistance genes.

The increase demand for fresh vegetables is causing an expansion of the market for minimally processed vegetables along with new recognized food safety problems. To gain further insight on this topic we analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying acquired antibiotic resistance genes, food products scarcely considered in surveillance studies. A total of 50 RTS (7 brands; split or mixed leaves, carrot, corn) were collected in 5 national supermarket chains in Porto region (2010). They were tested for aerobic mesophilic counts, coliforms and Escherichia coli counts as well as for the presence of Salmonella and Listeria monocytogenes. Samples were also plated in different selective media with/without antibiotics before and after enrichment. The E. coli, other coliforms and Enterococcus recovered were characterized for antibiotic resistance profiles and clonality with phenotypic and genetic approaches. A high number of RTS presented poor microbiological quality (86%--aerobic mesophilic counts, 74%--coliforms, 4%--E. coli), despite the absence of screened pathogens. In addition, a high diversity of bacteria (species and clones) and antibiotic resistance backgrounds (phenotypes and genotypes) were observed, mostly with enrichment and antibiotic selective media. E. coli was detected in 13 samples (n=78; all types and 4 brands; phylogenetic groups A, B1 and D; none STEC) with resistance to tetracycline [72%; tet(A) and/or tet(B)], streptomycin (58%; aadA and/or strA-strB), sulfamethoxazole (50%; sul1 and/or sul2), trimethoprim (50%; dfrA1 or dfrA12), ampicillin (49%; blaTEM), nalidixic acid (36%), ciprofloxacin (5%) or chloramphenicol (3%; catA). E. coli clones, including the widespread group D/ST69, were detected in different samples from the same brand or different brands pointing out to a potential cross-contamination. Other clinically relevant resistance genes were detected in 2 Raoultella terrigena carrying a bla(SHV-2) and 1 Citrobacter freundii isolate with a qnrB9 gene. Among Enterococcus (n=108; 35 samples; Enterococcus casseliflavus--40, Enterococcus faecalis--20, Enterococcus faecium--18, Enterococcus hirae--9, Enterococcus gallinarum--5, and Enterococcus spp.--16) resistance was detected for tetracyclines [6%; tet(M) and/or tet(L)], erythromycin [3%; erm(B)], nitrofurantoin (1%) or ciprofloxacin (1%). The present study places ready-to-eat salads within the spectrum of ecological niches that may be vehicles for antibiotic resistance bacteria/genes with clinical interest (e.g. E. coli-D-ST69; bla(SHV-2)) and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.

Characterisation of plasmids implicated in the mobilisation of extended-spectrum and AmpC ß-lactamase genes in clinical Salmonella enterica isolates and temporal stability of the resistance genotype.

Plasmids implicated in the mobilisation of ß-lactamase genes in extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Salmonella enterica isolates recovered from three Spanish hospitals were characterised. The temporal stability of these plasmids and of the resistance phenotype without antimicrobial pressure was also assessed in the laboratory setting. The resistance determinants and their genetic environments were characterised by PCR sequencing, and their genomic location was analysed by S1 nuclease pulsed-field gel electrophoresis (PFGE) and I-CeuI PFGE, followed by Southern blot hybridisation. The 11 S. enterica studied strains carried blaCTX-M-9 (serovar Virchow, 2 isolates), blaCTX-M-10 (Virchow, 2), blaCTX-M-14 (Enteritidis, 1), blaCTX-M-15 (Gnesta and S. enterica group C, 2), blaSHV-2 (Livingstone, 1), blaSHV-12 (Enteritidis, 1) and blaCMY-2 (Bredeney, 2). The ISEcp1-blaCTX-M-14-IS903 and ISEcp1-blaCTX-M-15-orf477 genetic structures were detected. IncI1 and IncA/C plasmids carried blaCTX-M-14, blaCTX-M-15, blaSHV-2, blaSHV-12 and blaCMY-2 genes. blaCTX-M-9 included in an In60 complex integron and blaCTX-M-10 linked to a phage-related element were found in non-typeable plasmids. Conjugation and temporal stability experiments were performed in vitro through daily passages (100 days) in the absence of antimicrobial pressure. In the stability experiments, 5 of the 11 tested isolates lost the ESBL or AmpC plasmidic genes and this was associated with concomitant loss of the whole or partial plasmid. In conclusion, successful plasmids belonging to different Inc groups mobilise ESBL- and AmpC-encoding genes in S. enterica. Loss of ESBL/AmpC genes in the absence of antimicrobial pressure might explain the low prevalence of these ß-lactamases among Salmonella isolates.