Bla Gene Research Articles

Variants of the gentamicin and tobramycin resistance plasmid pRAY are widely distributed in Acinetobacter

To determine the cause of resistance to the aminoglycosides gentamicin and tobramycin in Acinetobacter isolates and the location of the resistance genes. Australian Acinetobacter baumannii isolates were screened for resistance to aminoglycosides. PCR followed by restriction digestion of amplicons was used to detect genes and plasmids. Plasmids were isolated and examined by restriction digestion. Plasmid DNA sequences were determined and bioinformatic analysis was used to identify features. The sequence of the bla(OXA-Ab) gene and multilocus sequence typing were used to determine strain types. Isolates that exhibited resistance to gentamicin, kanamycin and tobramycin were of diverse strain types. These isolates all carried the aadB gene cassette, and in all but one the cassette was in a 6 kb plasmid similar to pRAY. The three plasmid sequences determined revealed multiple frame-shift differences in the available pRAY sequence that altered the reading frames. In pRAY*, mobA and mobC mobilization genes were identified, but no potential replication initiation protein was found. pRAY*-v1 differed from pRAY* by 66 single-base differences, and pRAY*-v2 included two insertion sequences, ISAba22, located upstream of the aadB gene cassette, and IS18-like, within ISAba22. The plasmid pRAY* and variants are widely distributed in Acinetobacter spp. and are the most common cause of resistance to gentamicin and tobramycin. Mobilization genes should assist in the dissemination of pRAY* and its variants.

Development and validation of a multiplex TaqMan real-time PCR for rapid detection of genes encoding four types of class D carbapenemase in Acinetobacter baumannii

A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D β-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87 %) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2 %) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.

Increased raw poultry meat colonization by extended spectrum beta-lactamase-producing Escherichia coli in the south of Spain

The present study was conducted to assess the prevalence of retail chicken and turkey meat colonized by extended-spectrum beta-lactamase-producing Escherichia coli (ESBLEC) in Seville, Spain. ESBLEC recovered from meat samples purchased in 2010 were characterized by specific PCR analysis for bla genes, phylogenetic groups and subgroups (genotypes) and O25b/pabB/B2 traits of ST131. Results were compared with those obtained in a previous study in 2007, when a high percentage of retail meat samples were found to be colonized by ESBLEC. The prevalence of retail poultry meat colonized by ESBLEC increased from 62.5% in 2007 to 93.3% in 2010 (p=0.005). Non-pathogenic B1 and A1 genotypes accounted for more than 60% of the 60 isolates recovered. Sequence type ST131 or B2 phylogroup isolates were not detected. Clonal relatedness was detected in just 2 CTX-M-1-producing isolates from 2 chicken samples belonging to phylogenetic group A, genotype A1. There continued to be a significantly high quinolone resistance, with 85.4% and 32.2% of isolates showing resistance to nalidixic acid and ciprofloxacin, respectively. SHV-12 was the most common ESBL harbored by E. coli, although it has decreased in prevalence since 2007. Meanwhile, CTX-M ESBLs prevalence has increased. We conclude that the trend of colonization by ESBLECs—particularly CTX-M-producing isolates—in raw poultry meat has increased in a short period of time in our area.

High prevalence of extended-spectrum- -lactamase-producing Enterobacteriaceae in organic and conventional retail chicken meat, Germany

To determine the prevalence of extended-spectrum β-lactamase (ESBL) production in Enterobacteriaceae in retail chicken meat in Germany. A total of 399 chicken meat samples from nine supermarket chains, four organic food stores and one butcher's shop in two geographically distinct regions (Berlin and Greifswald) were screened for ESBL production using selective agar. Phenotypic ESBL isolates were tested for bla(TEM), bla(CTX-M) and bla(SHV) genes using PCR and DNA sequencing. Antibiotic coresistances were determined and strain typing was performed using PCR-based phylogenetic grouping and XbaI-PFGE. A total of 185 confirmed ESBL isolates were obtained from 175 samples (43.9%) from all tested sources. The majority of isolates were Escherichia coli producing ESBL types SHV-12 (n = 82), CTX-M-1 (n = 77) and TEM-52 (n = 16). No differences could be observed in the prevalence of ESBL producers between organic and conventional samples. 73.0% of the ESBL producers showed coresistance to tetracycline, 35.7% to co-trimoxazole and 7.6% to ciprofloxacin. Strain typing of selected E. coli isolates from Berlin revealed identical macrorestriction patterns for several isolates from samples taken from the same stores. This is the first comprehensive study from Germany showing a high prevalence of TEM-, CTX-M- and SHV-type ESBLs in Enterobacteriaceae isolated from retail chicken meat. The high rate of coresistance to different classes of antibiotics in the ESBL producers might reflect the common veterinary usage of these and related substances. There is an urgent need to further evaluate the role of poultry in the transmission of highly resistant ESBL-producing bacteria in humans.

Role of ISAba1 and ISAba125 in governing the expression of bla ADC in clinically relevant Acinetobacter baumannii strains resistant to cephalosporins

Acinetobacter baumannii is a multi-resistant opportunistic nosocomial pathogen responsible for several outbreaks worldwide. It can cause several infections at various sites of the body. One of the main infections caused by this bacterium is ventilator-associated pneumonia in patients in intensive care units. Treating these infections is becoming difficult because of the high resistance to antimicrobial agents. This study compared the expression of the chromosomally encoded bla(ADC) gene in isolates having ISAba1, ISAba125 and no insertion upstream of the bla(ADC) gene in A. baumannii clinical isolates. It showed that the expression of bla(ADC) was six times greater when ISAba125 was present upstream of the gene in comparison with the constitutively expressed bla(ADC) gene with no insertion present upstream. The study indicated that ISAba125 has better promoters than ISAba1 and this is responsible for the overexpression of the bla(ADC) gene as they share considerable homology to the well-established Escherichia coli promoters. The -10 box of ISAba125 formed a fusion promoter with the -35 box of the bla(ADC) gene causing the bla(ADC) gene to be significantly overexpressed. The ability to upregulate the expression of bla(ADC) with the assistance of different insertion elements such as ISAba1 and ISAba125 has become an important factor in A. baumannii resistance to cephalosporins.

Charakterisierung der extended spectrum b-Lactamasen (ESBL) und Bestimmung der von Kindern isolierten uropathogenen Virulenzfaktoren von Escherichia coli (E. coli) Stämmen

The aim of the study was to characterize ESBL-producing uropathogenic Escherichia coli (UPEC) strains isolated in children. That included the investigation of virulence factors and the analysis of the types of β-lactamases at the molecular genetic level. During the 2-year study period, 77 ESBL-producing E. coli strains were recovered from urine samples of febrile children with significant bacteriuria hospitalized at one Croatian hospital. Susceptibility of isolates to bactericidal serum activity was tested by Shiller and Hatch method, while adhesin expression was determined by agglutination methods. Characterization of ESBLs was performed by PCR with specific primers for ESBLs and by sequencing of bla (ESBL) genes. Genotyping of the E. coli isolates was performed by pulsed-field gel electrophoresis (PFGE). Twenty-seven (35.1%) and 50 (64.9%) ESBL-producing UPEC strains were isolated in neonates and infants, respectively. Of 70 strains investigated for the presence of virulence factors, adhesins were detected in 48.6% strains (8.6% in the neonate and 40% in the infants group) giving a statistically significant difference in adhesin expression between the two groups (p < 0.01). Hemolysin was produced by 84.3%, whereas 70% of strains were serum-resistant. The bla (TEM) gene was detected in 22 (28%) and bla (SHV) gene in 57 strains (74%), whereas bla (CTX-M) gene was detected in only two isolates (2.5%). In ten isolates, bla (TEM) and bla (SHV) were simultaneously detected. Sequencing of bla (SHV) genes revealed that SHV-5 β-lactamase was by far the most prevalent and was found in 51 strains (66%). The strains were clonally related as demonstrated by PFGE and assigned into ten clusters. Infection control measures should be employed and the consumption of expanded-spectrum cephalosporins in the hospital should be restricted.

Study on the Resistant Genes to Carbapenems and Epidemiological Characterization of Multidrug-ResistantAcinetobacter baumanniiIsolates

The purpose of this study was to examine the carbapenemase-encoding resistance genes and analyze homologous of multidrug-resistant Acinetobacter baumannii (MRAB) isolates from nosocomial infections. Seventy-six A. baumannii strains were collected from inpatients and object surface of devices in intensive care units from May 2008 to February 2011. Antibiotic susceptibility testing of 18 antimicrobial agents was performed. The presence of carbapenemase-encoding resistance genes was investigated by polymerase chain reaction. Genotyping and dendrogram analysis of A. baumannii strains from nosocomial infections were performed using the DiversiLab System. All of the 76 clinical A. baumannii isolates were shown multidrug resistance. The bla(OXA-23) gene was identified in the 76 MRAB strains, while bla(OXA-24), bla(OXA-58), VIM, IMP-1, IMP-4, SIM, and blaNDM-1 were absent in all. Twenty-four A. baumannii strains from the samples with nosocomial infections were classified into four unrelated groups and nine patterns. In conclusion, production of bla(OXA-23) in MRAB is one of the molecular mechanisms responsible for carbapenem resistance. The MRAB strains from unrelated groups show different drug resistance, but the homologous strains also have different drug resistance. Homologous analysis can provide scientific evidence for evaluation of epidemic status of nosocomial infection caused by MRAB.

Prevalence and characteristics of -lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China

To determine the molecular epidemiology of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) in Escherichia coli isolated from farmed fish in China. E. coli was isolated from fish gut samples from fish farmed throughout Guangdong province and tested for the presence of the β-lactamase genes and PMQR-encoding genes using PCR and DNA sequence analysis. Co-transfer of plasmids encoding for ESBLs as well as PMQR determinants was explored by conjugation into E. coli. A total of 218 non-duplicate E. coli were recovered from fish gut samples. β-Lactamase genes were identified in 19 (17%) of 112 strains with reduced susceptibility to ampicillin, and PMQR genes were identified in 59 (73.8%) of 80 strains with reduced susceptibility to ciprofloxacin. Only three ESBL genes were identified in three isolates: bla(CTX-M-14), bla(CTX-M-79) and bla(SHV-27). PMQR gene screening identified qnr genes (n = 59) as the most common, including qnrB (n = 33), qnrS (n = 21) and qnrD (n = 5), with aac(6')-Ib-cr (n = 6) being rarely found. The co-carriage of two or three PMQR genes in one strain was found in 7 (11.9%) isolates. The ESBL gene bla(CTX-M-79) was found to be co-carried with qnrS. Co-transfer of qnrS was observed with bla(CTX-M-79). Our study is the first to demonstrate the existence of high levels of mobile genes conferring reduced susceptibility to fluoroquinolones as well as the presence of ESBL genes in fish produced in China, and identifies a significant reservoir of antibiotic resistance genes relevant to human medicine.

Co-production of SFO-1 and DHA-1 -lactamases and 16S rRNA methylase ArmA in clinical isolates of Klebsiella pneumoniae

To investigate the epidemiology and genetic characteristics of the plasmid-mediated extended-spectrum β-lactamase (ESBL) gene bla(SFO-1) in clinical isolates of ESBL-producing Klebsiella pneumoniae and Escherichia coli. The prevalence of the bla(SFO-1) gene was examined by PCR amplification. Conjugation and transformation experiments were performed and the presence of antimicrobial resistance determinants was investigated by the PCR method. The genetic environments of the bla(SFO-1) and armA genes were determined by direct sequencing of plasmid pHS20. Plasmids were typed by PCR-based replicon typing. PFGE and multilocus sequence typing were performed on SFO-producing strains. Of 158 ESBL-producing strains of K. pneumoniae, 3 (1.9%) carried the bla(SFO-1) gene. All of the SFO-producing isolates belonged to the ST11 epidemic clone, with a single PFGE type and had high-level resistance to third-generation cephalosporins, aminoglycosides and ciprofloxacin. The bla(SFO-1) gene was co-transferred with the armA, aac(6')-Ib-cr and bla(TEM-1) genes by transformation, whereas the armA, bla(DHA-1) and qnrB4 genes were co-transferred by conjugation. The armA genes were located within the composite transposon Tn1548 on two different plasmids in the strain 08-129. The bla(SFO-1) gene was located upstream of an ampR gene from the coding region and flanked by two inverted repeats of IS26. Plasmids carrying bla(DHA-1) were identified as IncFII, while the bla(SFO-1)-bearing plasmids were non-typeable. Although SFO-1 is a low-occurrence ESBL, it has been captured by a plasmid accumulating multiple resistance determinants including armA and aac(6')-Ib-cr, and accompanied by a large DHA-1-bearing IncFII plasmid in a prevalent K. pneumoniae ST11 clone.

Proteomic Analysis of Clinical Isolate of Stenotrophomonas maltophilia with blaNDM-1,blaL1 and blaL2 β-Lactamase Genes under Imipenem Treatment

The co-occurrence of L1 and AmpR-L2 with bla(NDM-1) gene with an upstream 250-bp promoter was detected in a clinical isolate of Stenotrophomonas maltophilia DCPS-01, which was resistant to all β-lactams and sensitive only to colistin and fluoroquinolones. To investigate expression of resistance genes and the molecular mechanisms of bacteria resistance to carbapenems, proteomic profiles of the isolate was passaged with and without the drug by using 2D-PAGE. The results showed that 33 genes exhibiting a ≥3-fold change were identified as candidates that may help S. maltophilia survive drug selection. Strikingly, L1 was expressed more highly in cells grown with imipenem, and the abundant NDM-1 further increased, while very little L2 was detected even following induction. Specific activities for β-lactamase revealed that L2 remained at constitutive low levels (10.6 U/mg), while L1 and NDM-1 showed clear activity (69.8 U/mg). Our data support that imipenem could specifically and reversibly induce L1 and NDM-1, which together played key roles in drug resistance in DCPS-01. Although NDM-1 mediated resistance to carbapenems has been found in very few cases, to our knowledge, this is the first proteomics research of S. maltophilia with NDM-1, giving very broad-spectrum antibiotic resistance profiles.

Commensal Pseudomonas Species Isolated from Wastewater and Freshwater Milieus in the Eastern Cape Province, South Africa, as Reservoir of Antibiotic Resistant Determinants

Pseudomonas species are opportunistic pathogens with implications in a wide range of diseases including cystic fibrosis and sickle cell anaemia. Because of their status as multidrug resistant (MDR) and extremely drug resistant (XDR) bacteria Pseudomonas species represent a threat to public health. Prevalence, antibiogram and associated antibiotic resistant genes of Pseudomonas species isolated from freshwater and mixed liquor environments in the Eastern Cape Province of South Africa were assessed. Polymerase chain reaction (PCR) based technique was used to identify the isolates and screen for antibiotic resistant genes. The result shows occurrence of Pseudomonas spp. in freshwater and mixed liquor as follows: 71.42% and 37.5% (P. putida), 14.28% and 31.25% (P. flourescens), 7.14% and 6.25% (P. aeruginosa) and 7.14% and 25% for other Pseudomonas species respectively. Disk diffusion antibiogram of the Pseudomonas isolates from the two locations showed 100% resistance to penicillin, oxacillin, clindamycin, rifampicin and 100% susceptibility to ciprofloxacin and gentamicin with varied percentage resistances to cephalothin, nalidixic acid, tetracycline, and ampicillin. The blaTEM antibiotic resistant gene was detected in 12.5% of P. putida, 57.14% of P. fluorescens, 100% P. aeruginosa and 40% in other Pseudomonas species. Similarly, Integrons conserved segment were detected in 12.5% of P. putida, 57.14% of P. fluorescens, 100% of P. aeruginosa and 40% of other Pseudomonas species. The presence of blaTEM gene and integrons conserved segment in some of the isolates is worrisome and suggest Pseudomonas species as important reservoirs of multidrug resistance genes in the Eastern Cape Province environment.

Frequent detection of plasmid-mediated quinolone resistance (qnr) genes in multidrug-resistant Enterobacteriaceae blood isolates, Hong Kong

Plasmid-mediated quinolone resistance (qnr) genes confer low-level resistance but provide background for selection of highly-resistant strains. We investigated their prevalence and significance in clinical Enterobacteriaceae bacteremic isolates in Hong Kong. A prospective, hospital-based study was conducted (January 2008 to March 2009). Consecutive, non-duplicate blood isolates of extended-spectrum-β-lactamase (ESBL) and/or plasmid-mediated AmpC (PMAmpC) β-lactamase-producing Enterobacteriaceae were collected and subjected to qnr genes detection using multiplex PCR. Direct sequencing was performed to characterize the qnr and the co-existing bla genes. Clinical and microbiological variables, including antimicrobial resistance profiles, were compared between infections by 'qnr-positive' and 'qnr-negative' Enterobacteriaceae. Altogether 199 ESBL/PMAmpC-producing Enterobacteriaceae isolates were studied. qnr genes were detected in 20 % (qnrB, n = 24; qnrS, n = 16; qnrA, n = 0), of which 85% were Klebsiella spp. There was a strong association with PMAmpC genes (qnrB and DHA-1; p < 0.001). 'qnr-positive' isolates were more commonly hospital-acquired (60.0% vs 35.8%; adjusted OR 2.68, 95%CI 1.32-5.46) and multidrug-resistant (e.g. amoxicillin-clavulanate 90-100%, piperacillin-tazobactam 40-57%, ceftazidime 53-78%; sulfamethoxazole/trimethoprim 60-70%; ciprofloxacin 53-65%, levofloxacin 35-48%). Patients with 'qnr-positive' Enterobacteriaceae bacteremia had a higher 30-day mortality (45% vs 22%, p = 0.003). High Pitt bacteremia score, development of pneumonia, and failure to receive susceptible fluoroquinolone (adjusted HR 4.27; 95%CI 1.45-12.61) or carbapenem (adjusted HR 3.04; 95%CI 1.49-6.20) treatment were independent factors associated with death. A high proportion of ESBL/PMAmpC-producing Enterobacteriaceae (typically Klebsiella) bacteremic isolates carried qnr. These strains were multidrug-resistant, which was associated with inappropriate treatment and high fatality. Further dissemination of qnr and selection of fluoroquinolone/β-lactam-resistant strains should be closely monitored and controlled.

Epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae in a UK district hospital; an observational study

Extended-spectrum beta-lactamases (ESBLs) are an increasingly important cause of resistance in Gram-negative bacteria throughout the world. We investigated the clinical and molecular epidemiology of infections caused by ESBL-producing Enterobacteriaceae in a UK hospital, to identify the types of ESBL produced and risk factors for acquisition. Between July 2008 and June 2009, all patients yielding ESBL-producing Enterobacteriaceae from any clinical specimen were prospectively investigated using a questionnaire. API20E was used for bacterial identification; susceptibility testing and ESBL production were assessed by BSAC disc diffusion and cefpodoxime-clavulanate synergy tests, respectively. Polymerase chain reaction was used to screen a subset of isolates for bla(CTX-M) genes, to assign Escherichia coli isolates to their phylogenetic groups, and to identify members of the uropathogenic ST131 lineage. The overall prevalence of ESBL producers among clinical samples yielding Enterobacteriaceae was 1%; ESBL producers, obtained from 124 patients, were E.coli (N=105), Klebsiella pneumoniae (N=12), and others (N=7). The main risk factors identified include recent antibiotic use (93%) and presence of a urinary catheter (24%). CTX-M group 1 ESBLs dominated (in 59 of 78, 76%, isolates studied). Most E.coli (35 of 56 tested) were phylogroup B2; of these, 23 belonged to the ST131 clone, 12 were phylogroup D, and four each belonged to phylogroups A and B1. ESBLs are an uncommon but significant problem in north-west Cambridgeshire. CTX-M-type enzymes were found in 75% of ESBL-positive isolates. All but two patients had at least one recognized risk factor. This study supports the requirement for interventions to reduce inappropriate urinary catheterization and antibiotic prescribing.

Mechanisms of resistance to cephalosporin and emergence of O25b‐ST131 clone harboring CTX‐M‐27 β‐lactamase in extraintestinal pathogenic Escherichia coli from dogs and cats in Japan

Thirty-three cefazolin-resistant extraintestinal pathogenic Escherichia coli strains from companion animals were screened for bla(CMY-1) , bla(CMY-2) , bla(SHV) , bla(TEM) , and bla(CTX-M) genes. Extended-spectrum β-lactamase-producing strains were further characterized by O serotyping and multilocus sequence typing. It was found that 20 and 17 isolates harbored TEM-1 and CMY-2 β-lactamases, respectively, and 13 isolates harbored both β-lactamases. One isolate harbored DHA-1 β-lactamase. Eleven isolates were found to possess CTX-M β-lactamases (CTX-M-27 [n= 6], CTX-M-14 [n= 3], CTX-M-15 [n= 1], and CTX-M-55 [n= 1]). Of 11 CTX-M-positive strains, four strains were O25b-ST131 clones harboring CTX-M-27, and the remaining seven strains belonged to O6-ST127, ONT-ST354, O159-ST539, O1-ST648, O8-ST1642, O25b-ST2042, and ONT-ST2178.

Antimicrobial susceptibilities and resistance genes in Campylobacter strains isolated from poultry and pigs in Australia

To evaluate the phenotypic and genotypic profiles of Campylobacter spp. from poultry faecal samples from free range or intensively raised meat chickens and free range egg layers. In addition, a case-comparison study of antibiotic resistance genes from different groups of poultry and some pig strains previously collected was carried out. Resistance to different antibiotics was assessed using the agar dilution method. In addition, all the strains were tested for ampicillin (bla(OXA-61) ), erythromycin (aph-3-1), tetracycline tet(O), streptomycin (aadE), and the energy-dependent multi-drug efflux pump (cmeB) resistance genes using multiplex polymerase chain reaction. The evaluation of phenotypic resistance revealed all of the strains from poultry were sensitive to ciprofloxacin, gentamicin, erythromycin or tylosin. But, widespread resistance to lincomycin (51-100%), extensive resistance to ampicillin (33·3-60·2%) and less resistance to tetracycline (5·6-40·7%) were observed in the different groups of chickens. Antibiotic resistance genes bla(OXA-61,) cmeB and tet(O) were found in 82·6-92·7%, 80·3-89% and 22·3-30·9% Camp.coli isolates from pigs, whilst 59-65·4% and 19·2-40·7% Camp.jejuni from chickens were found to encode bla(OXA-61) and tet(O), respectively. No significant difference between isolates from free range egg layers and meat chickens (P<0·05) was found. However, there were significant differences between the pig strains and all the groups of poultry strains (P<0·05) with regard to carriage of resistance genes. In addition, pulsed field gel electrophoresis of selected resistant isolates from the poultry and pig revealed closely related clonal groups. Our results suggest the resistant strains are persisting environmental isolates that have been acquired by the different livestock species. Furthermore, the different treatment practices in poultry and pigs have resulted in differences in resistance profiles in Campylobacter isolates.

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

Pathogenic Klebsiella pneumoniae, resistant to beta-lactam and quinolone drugs, is widely recognized as important bacteria causing array of diseases. The resistance property is obtained by acquisition of plasmid encoded blaTEM, blaSHV, blaCTX-M, QNRA, QNRB and QNRS genes. The aim of this study was to document the prevalence and association of these resistant genes in K. pneumoniae infecting patients in India. Approximately 97 and 76.7% of the 73 K. pneumoniae isolates showed resistance towards beta-lactam and quinolone drugs respectively. Bla genes were detected in 74% of K. pneumoniae isolates; with prevalence in the following order: blaTEM>blaSHV>blaCTXM. QNR genes were detected in 67% samples. Chi-square analysis revealed significant association between presence of bla and qnr genes in our study (P value=0.000125). Sequence analysis of some blaTEM, blaSHV, blaCTX-M and QNRB PCR products revealed presence of blaTEM1 (GenBank accession: JN193522), blaTEM116 (JN193523 and JN193524), blaSHV11, blaCTXM72 variants (JF523199) and QNRB1 (JN193526 and JN193527) in our samples.

Carbapenem resistance and Acinetobacter baumannii in Senegal: the paradigm of a common phenomenon in natural reservoirs.

Incidence of carbapenem-resistant Acinetobacter baumannii is rising in several parts of the world. In Africa, data concerning this species and its resistance to carbapenems are limited. The objective of the present study was to identify the presence of A. baumannii carbapenem-resistant encoding genes in natural reservoirs in Senegal, where antibiotic pressure is believed to be low. From October 2010 to January 2011, 354 human head lice, 717 human fecal samples and 118 animal fecal samples were screened for the presence of A. baumannii by real time PCR targeting bla OXA51-like gene. For all samples positive for A. baumannii, the carbapenemase-hydrolysing oxacillinases blaOXA23-like and blaOXA24-like were searched for and sequenced, and the isolates harbouring an oxacillinase were genotyped using PCR amplification and sequencing of recA gene. The presence of A. baumannii was detected in 4.0% of the head lice, in 5.4% of the human stool samples and in 5.1% of the animal stool samples tested. No bla OXA24 gene was detected but six fecal samples and three lice were positive for bla OXA23-like gene. The bla OXA23-like gene isolated in lice was likely a new oxacillinase sequence. Finally, the A. baumannii detected in stools were all of recA genotype 3 and those detected in lice, of recA genotype 4. This study shows for the first time a reservoir of bla OXA23-like-positive gene in human head lice and stool samples in Senegal.

Transmission Dynamics of Extended-Spectrum β-lactamase–Producing Enterobacteriaceae in the Tertiary Care Hospital and the Household Setting

Studies about transmission rates of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in hospitals and households are scarce. Eighty-two index patients with new carriage of ESBL-producing Escherichia coli (ESBL-Ec; n = 72) or ESBL-producing Klebsiella pneumoniae (ESBL-Kp; n = 10) and their hospital (n = 112) and household (n = 96) contacts were studied prospectively from May 2008 through September 2010. Isolates were phenotypically and molecularly characterized (sequencing of bla genes, repetitive extragenic palindromic polymerase chain reaction, pulse-field gel electrophoresis, and multilocus sequence typing). Transmission was defined as carriage of a clonally-related ESBL producer with identical bla(ESBL) gene(s) in the index patient and his or her contact(s). CTX-M-15 was the most prevalent ESBL in ESBL-Ec (58%) and ESBL-Kp (70%) in the index patients. Twenty (28%) ESBL-Ec isolates were of the hyperepidemic clone ST131. In the hospital, transmission rates were 4.5% (ESBL-Ec) and 8.3% (ESBL-Kp) and the incidences of transmissions were 5.6 (Ec) and 13.9 (Kp) per 1000 exposure days, respectively. Incidence of ESBL-Kp hospital transmission was significantly higher than that of ESBL-Ec (P < .0001), despite implementation of infection control measures in 75% of ESBL-Kp index patients but only 22% of ESBL-Ec index patients. Detection of ESBL producers not linked to an index patient was as frequent (ESBL-Ec, 5.7%; ESBL-Kp, 16.7%) as nosocomial transmission events. In households, transmission rates were 23% for ESBL-Ec and 25% for ESBL-Kp. Household outweighs nosocomial transmission of ESBL producers. The effect of hospital infection control measures may differ between different species and clones of ESBL producers.

Characterization of Carbapenemase Genes in Enterobacteriaceae Species Exhibiting Decreased Susceptibility to Carbapenems in a University Hospital in Chongqing, China

BackgroundOur study was to investigate the prevalence of carbapenemase genes in strains of Enterobacteriaceae species exhibiting decreased susceptibility to carbapenems in our hospital.MethodsThe carbapenemase producing Enterobacteriaceae species were confirmed by modified Hodge test (MHT) and EDTA-disc synergy test which indicating the production of class B carbapenemases. PCR and sequencing analysis were used to identify the drug-resistant genes. DNA fingerprinting based on enterobacterial repetitive intergenic consensus (ERIC)-PCR was applied to investigate the homology of Enterobacteriaceae species.ResultsFrom a collection of 1,472 Enterobacteriaceae species, 18 isolates with decreased susceptibility to carbapenem treatment were identified and 9 of which were positive by MHT, and 6 of which produced class B carbapenemases. PCR and sequencing analysis of the 18 isolates revealed 4 different carbapenemase genes (blaIMP-8, blaoxa-1, blaIMP-26, and blaoxa-47) in 10 isolates, with the blaIMP-8 and blaoxa-1 genes being the most common (60-70% prevalence). ERIC-PCR showed 5, 2, and 2 unique genotypes for Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae, respectively. Three E. coli strains isolated from different patients from the urologic surgery department exhibited the same DNA banding pattern, suggesting a possible clonal dissemination. Majority (17/18) of the carbapenem-unsusceptible Enterobacteriaceae species isolates was obtained from the surgery department of our hospital.ConclusionsThe main carbapenemase genes of Enterobacteriaceae species in our hospital were blaIMP-8 and blaoxa-1. Prevalence of carbapenem resistance may be existed in surgery department and infection control should be taken for preventing further dissemination of drug-resistant strains.

Presence of extended-spectrum beta-lactamase-producing Enterobacteriaceae in waste waters, Kinshasa, the Democratic Republic of the Congo

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are a major public health concern. We previously demonstrated the presence of ESBL-producing Enterobacteriaceae in sachet-packaged water bags sold in Kinshasa, the Democratic Republic of the Congo. In complement to the previous study, we aimed to assess the presence of ESBL-producing Enterobacteriaceae in waste waters in Kinshasa.Enterobacteriaceae isolates recovered from environmental water samples were screened and phenotypically confirmed as ESBL-producers by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI M100-S21). Final identification to the species level and further antimicrobial susceptibility testing were carried out with MicroScan® NBC42 panels and the identification of bla (ESBL) coding genes was performed by a commercial multiplex ligation polymerase chain reaction (PCR) microarray (Check-Points CT 101, Wageningen, the Netherlands). Overall, 194 non-duplicate Enterobacteriaceae were recovered from several sewer and river sites in nine out of 24 municipalities of Kinshasa. Fourteen isolates (7.4%) were confirmed as ESBL-producers, the main species being Enterobacter cloacae (46.6%) and Klebsiella pneumoniae (40.0%). Associated resistance to both aminoglycoside and fluoroquinolone antibiotics was observed in ten isolates; the remaining isolates showed co-resistance to either fluoroquinolone (n = 3) or to aminoglycoside (n = 1) alone. All but one isolate carried bla (CTX-M) genes belonging to the CTX-M-1 group. ESBL-producing Enterobacteriaceae are increasingly being reported from various sources in the community. The present results suggest that ESBL-producing Enterobacteriaceae are widespread in the environment in the community of Kinshasa. Cities in Central Africa should be added to the map of potentially ESBL-contaminated environments and highlight the need to reinforce safe water supply and public sanitation.