Draft Genome Sequence of a Multidrug-Resistant Acinetobacter baumannii Strain from Chile.

Acinetobacter baumannii strain Ab5 was isolated in the year 2007 in Chile, being one of the first multidrug-resistant (MDR) cases reported in the country. Here, we present the very first draft genome sequence of an MDR Chilean strain, which shows the presence of diverse resistance and acquired virulence genes.

Dissemination of multiple carbapenem-resistant clones of Acinetobacter baumannii in the Eastern District of Saudi Arabia.

It has previously been shown that carbapenem-resistant Acinetobacter baumannii are frequently detected in Saudi Arabia. The present study aimed to identify the epidemiology and distribution of antibiotic resistance determinants in these bacteria. A total of 83 A. baumannii isolates were typed by pulsed-field gel electrophoresis (PFGE), and screened by PCR for carbapenemase genes and insertion sequences. Antibiotic sensitivity to imipenem, meropenem, tigecycline, and colistin were determined. Eight different PFGE groups were identified, and were spread across multiple hospitals. Many of the PFGE groups contained isolates belonging to World-wide clone 2. Carbapenem resistance or intermediate resistance was detected in 69% of isolates. The bla VIM gene was detected in 94% of isolates, while bla OXA-23-like genes were detected in 58%. The data demonstrate the co-existence and wide distribution of a number of clones of carbapenem-resistant A. baumannii carrying multiple carbapenem-resistance determinants within hospitals in the Eastern Region of Saudi Arabia.

Variations in IS6 promoters alter the expression of carbapenem resistance in related strains of Acinetobacter baumannii.

The aim of this work was to investigate the role of the IS6 family of insertion sequences present upstream of blaOXA-58 in two clonally related carbapenem-resistant Acinetobacter baumannii isolates obtained from paediatric cancer patients in Egypt. To determine their relatedness, the isolates were typed by pulsed-field gel electrophoresis (PFGE), and the intrinsic blaOXA-51-like gene was amplified and sequenced. Minimum inhibitory concentrations (MICs) to imipenem and meropenem was determined according to British Society of Antimicrobial Chemotherapy (BSAC) guidelines. PCR and sequencing of blaOXA-58 and the upstream and downstream regions was performed to determine the genetic environment. The two isolates were positive for the intrinsic blaOXA-64 gene, and the MICs for isolates AB-14298 and AB-P67 were 8mg/L and 64mg/L for imipenem and 2mg/L and 16mg/L for meropenem, respectively. The blaOXA-58 gene in AB-14298 was flanked by ISAba3 interrupted with IS1006, whereas AB-P67 had ISAba3 interrupted by IS1008, both belonging to the IS6 family of insertion sequences. In conclusion, both IS1006 and IS1008 provided suitable promoter sequences for expression of the downstream blaOXA-58 gene.

The genetic environment of the antiseptic resistance genes qacEΔ1 and cepA in Klebsiella pneumoniae.

The genetic environment of two antiseptic resistance genes (qacEΔ1 and cepA) was examined in Klebsiella pneumoniae isolates obtained from the Royal Infirmary of Edinburgh between 2006 and 2008. In 4 of 34 isolates, which carried the qacEΔ1 gene, the sul1 gene was located immediately downstream. In two of these, the orf5 gene of unknown function was found immediately downstream of the sul1 gene. In one case, this was substituted by the chrA gene. The cepA gene was carried by 56 (87·5%) isolates, and the pfkA gene was found directly downstream in 45 (70·3%) isolates, and in 40 (62·5%) of these isolates, the menG gene was found directly downstream. The cpxP gene was found in 47 (73·4%) isolates upstream of the cepA, and in 35 of these isolates, the cpxR gene was identified. These latter genes are transcription regulators, and reverse transcription polymerase chain reaction (RT-PCR) revealed that their presence was associated with cepA expression.

Clonal diversity of Acinetobacter baumannii from diabetic patients in Saudi Arabian hospitals.

Carbapenem-resistant Acinetobacter baumannii (CR-AB) represents a major health-care problem, causing high rates of morbidity and mortality. This study investigated the clonality of CR-AB isolated from diabetic patients from different regions in Saudi Arabia, as well as the relatedness of the ß-lactamase genes. A total of 64 non-repetitive CR-AB clinical isolates were collected from 16 different regions in Saudi Arabia from intensive care patients. Isolates were identified phenotypically by the Vitek 2 compact system and genotypically by amplification of the blaOXA-51-like gene. The target sequences were amplified by PCR and the clonal diversity of the isolates was explored by PFGE. Resistance studies revealed that the prevalence of imipenem and meropenem resistance was 92% and 96%, respectively, while the vast majority of the isolates were susceptible to tigecycline and colistin. In addition, blaVIM and blaOXA-23 were the most prevalent genes in the isolates under investigation, while ISAba1 was the most dominant insertion sequence. PFGE results showed 13 clusters; clone H was dominant, comprising 20 isolates from four hospitals, followed by clones C and F, comprising 11 isolates each from three and six hospitals, respectively. Moreover, the current study signified the clonal diversity of CR-AB in Saudi Arabia and showed the ability of some clones to infect patients in many different cities.

OXA ß-lactamases.

The OXA ß-lactamases were among the earliest ß-lactamases detected; however, these molecular class D ß-lactamases were originally relatively rare and always plasmid mediated. They had a substrate profile limited to the penicillins, but some became able to confer resistance to cephalosporins. From the 1980s onwards, isolates of Acinetobacter baumannii that were resistant to the carbapenems emerged, manifested by plasmid-encoded ß-lactamases (OXA-23, OXA-40, and OXA-58) categorized as OXA enzymes because of their sequence similarity to earlier OXA ß-lactamases. It was soon found that every A. baumannii strain possessed a chromosomally encoded OXA ß-lactamase (OXA-51-like), some of which could confer resistance to carbapenems when the genetic environment around the gene promoted its expression. Similarly, Acinetobacter species closely related to A. baumannii also possessed their own chromosomally encoded OXA ß-lactamases; some could be transferred to A. baumannii, and they formed the basis of transferable carbapenem resistance in this species. In some cases, the carbapenem-resistant OXA ß-lactamases (OXA-48) have migrated into the Enterobacteriaceae and are becoming a significant cause of carbapenem resistance. The emergence of OXA enzymes that can confer resistance to carbapenems, particularly in A. baumannii, has transformed these ß-lactamases from a minor hindrance into a major problem set to demote the clinical efficacy of the carbapenems.

Challenging the concept of bacteria subsisting on antibiotics.

Antibiotic resistance concerns have been compounded by a report that soil bacteria can catabolise antibiotics, i.e. break down and use them as a sole carbon source. To date this has not been verified or reproduced, therefore in this study soil bacteria were screened to verify and reproduce this hypothesis. Survival in high concentrations of antibiotics was initially observed; however, on further analysis these bacteria either did not degrade the antibiotics or they used an intrinsic resistance mechanism (ß-lactamases) to degrade the ß-lactams, as demonstrated by high-performance liquid chromatography. These results did not verify or reproduce the hypothesis that bacteria subsist on antibiotics or catabolise antibiotics as previously reported. This study identified that bacteria with a catabolising phenotype did not degrade streptomycin or trimethoprim and therefore could not utilise the antibiotics as a nutrient source. Therefore, we conclude that soil bacteria do not catabolise antibiotics.

Molecular diversity associated with the dissemination of CTX-M-15 beta-lactamase gene in blood culture isolates of Escherichia coli from Edinburgh.

BACKGROUND: Escherichia coli producing the CTX-M-15 ß-lactamase are a major cause of infection. We present the characterization of plasmids encoding the CTX-M-15 ß-lactamase gene, the genetic environment, and the mode of spread of this gene in blood culture isolates from a single hospital. METHODS: Blood culture E. coli isolates with extended spectrum ß-lactamase (ESBL) phenotype were screened for the presence of the bla(CTX-M) gene, other ESBLs, and aac(6')-Ib-cr genes. The genetic environment of bla(CTX-M) was determined by DNA sequencing. Plasmids were classified by their incompatibility group from polymerase chain reaction (PCR) replicon typing. Plasmid numbers and sizing were assessed by alkaline lysis and S1 nuclease digestion. Genotyping of the strains was determined by pulsed-field gel electrophoresis (PFGE) and ST131 by allele-specific PCR. RESULTS: Seven isolates had bla(CTX-M-15), with these isolates additionally having bla(TEM) (n = 5), bla(OXA) (n = 6), and aac(6')-Ib-cr (n = 6). Insertion sequence ISEcp1 was found upstream of the bla(CTX-M) gene, and in 2 isolates, ISEcp1 was found to be truncated with insertion sequence IS26. Plasmid replicon typing showed bla(CTX-M-15) genes were carried on the IncFII plasmid. All 7 isolates were associated with the O25b-ST131 clone. The PFGE banding pattern showed only 3 isolates were able to demonstrate clonality. CONCLUSIONS: This study shows the molecular diversity associated with the dissemination of bla(CTX-M-15) in a single Scottish hospital, which is largely due to horizontal transfer of multi- resistance IncF plasmids rather than clonal spread. It demonstrates that more detailed information is needed to monitor these bacteria to control them appropriately.

The rise of carbapenem-resistant Acinetobacter baumannii.

Acinetobacter spp. are Gram-negative bacteria that have become one of the most difficult pathogens to treat. The species A. baumannii, largely unknown 30 years ago, has risen to prominence particularly because of its ability to cause infections in immunocompromised patients. It is now a predominant pathogen in many hospitals as it has acquired resistance genes to virtually all antibiotics capable of treating Gram-negative bacteria, including the fluoroquinolones and the cephalosporins. Some members of the species have accumulated these resistance genes in large resistance islands, located in a "hot-spot" within the bacterial chromosome. The only conventional remaining treatment options were the carbapenems. However, A. baumannii possesses an inherent class D ß-lactamase gene (blaOXA-51-like) that can have the ability to confer carbapenem resistance. Additionally, mechanisms of carbapenem resistance have emerged that derive from the importation of the distantly related class D ß-lactamase genes blaOXA-23 and blaOXA-58. Although not inducible, the expression of these genes is controlled by mobile promoters carried on ISAba elements. It has also been found that other resistance genes including the chromosomal class C ß-lactamase genes conferring cephalosporin resistance are controlled in the same manner. Colistin is now considered to be the final drug capable of treating infections caused by carbapenem-resistant A. baumannii; however, strains are now being isolated that are resistant to this antibiotic as well. The increasing inability to treat infections caused by A. baumannii ensures that this pathogen more than ranks with MRSA or Clostridium difficile as a threat to modern medicine.

OXA-type carbapenemases in Acinetobacter baumannii in South America.

Acinetobacter baumannii is an opportunistic pathogen that is frequently involved in outbreaks of infection, occurring mostly in intensive care units. The increasing incidence of carbapenem resistance in A. baumannii worldwide is a concern since it limits drastically the range of therapeutic alternatives. The most important mechanism of carbapenem resistance is the enzymatic hydrolysis mediated by carbapenemases. In A. baumannii these enzymes are usually OXA-type carbapenemases, and belong to class D according to the classification of Ambler. The OXA-type carbapenemases are divided into five subgroups, four of which correspond to acquired carbapenemases, which accounts for the distribution of genes blaOXA in different geographic areas. In this work we review the different types of OXA-type carbapenemases present in A. baumannii, emphasizing the current situation in South America with special mention to the findings in Chile.

Plasmid-encoded PER-7 ß-lactamase responsible for ceftazidime resistance in Acinetobacter baumannii isolated in the United Arab Emirates.

OBJECTIVES: To investigate the mechanism of ceftazidime resistance in two isogenic Acinetobacter baumannii strains from the United Arab Emirates. METHODS: Two A. baumannii strains, NM55 and NM128, were isolated 4 months apart from a 6-year-old patient in the United Arab Emirates. Genotypic characterization was performed by PFGE and the MIC of ceftazidime was determined by the agar dilution method. Detection of bla(OXA) and metallo-ß-lactamase genes was performed by multiplex PCR. Analysis of bla(PER-7), ISAba1, bla(ADC) and the ISCR1 element was carried out by standard PCR. Plasmid analysis was achieved by Southern blotting. RESULTS: Strain NM55 was resistant to ceftazidime, whereas strain NM128 was susceptible. Both isolates carried the bla(OXA-23) and bla(OXA-64) genes and were identical according to their PFGE patterns. ISAba1 was present upstream of the bla(OXA-23) gene, but absent upstream of bla(ADC-26), in both strains. Strain NM55 possessed a bla(PER-7) gene with the presence of gst, a fragment of the abc transporter and a transposase gene downstream of it. The entire structure was part of an ISCR1 element and was located on an ≈ 200 kb plasmid in strain NM55, while the ceftazidime-susceptible NM128 strain carried an ≈ 180 kb plasmid without the bla(PER-7) gene. CONCLUSIONS: Ceftazidime resistance was mediated by a PER-7 ß-lactamase encoded in an ISCR1 element located on a plasmid. This represents the first detection of a PER-7 ß-lactamase encoded by a plasmid in A. baumannii.

Disruption of the blaOXA-51-like gene by ISAba16 and activation of the blaOXA-58 gene leading to carbapenem resistance in Acinetobacter baumannii Ab244.

OBJECTIVES: This study examines the mechanism of carbapenem resistance in Acinetobacter baumannii isolate Ab244. METHODS: A multiplex PCR for the detection of the bla(OXA-23-like), bla(OXA-40-like), bla(OXA-51-like) and bla(OXA-58-like) families was performed. MICs of imipenem and meropenem were determined by the agar dilution method. The sequence surrounding the bla(OXA-132) gene was determined by amplification with primer pairs encompassing a part of fxsA and an acetyltransferase gene (GNAT). The sequence upstream of the bla(OXA-58) gene was determined by sequencing. SDS-PAGE and carO PCR were performed to check the integrity of the outer membrane proteins. RT-PCRs for the expression of the bla(OXA-132) gene and the bla(OXA-58) gene were performed. RESULTS: Isolate Ab244 harboured bla(OXA-132) belonging to the bla(OXA-51-like) gene cluster and a bla(OXA-58) gene. The 4239 bp region between fxsA and GNAT showed an insert of ISAba16 (where IS stands for insertion sequence) after the first 15 nucleotides of the bla(OXA-132) gene, with an 8 bp target site duplication at the 5' and 3' ends of ISAba16. The sequence oriented in the 5'→3' direction caused insertional inactivation of the bla(OXA-132) gene. The bla(OXA-58) gene was highly expressed by the promoters provided by an ISAba3-like structure found upstream of the gene. The isolate was resistant to meropenem and had intermediate resistance to imipenem, and was also positive for ISAba1. CONCLUSIONS: This is the first report showing ISAba16-mediated inactivation of the bla(OXA-132) gene in strain Ab244. The resistance to carbapenems in strain Ab244 is related to the acquisition of the bla(OXA-58) gene, here governed by an ISAba3-like element.

Molecular epidemiology and antimicrobial resistance pattern of extended-spectrum-ß-lactamase-producing Enterobacteriaceae in Glasgow, Scotland.

OBJECTIVES: To establish the molecular epidemiology and antimicrobial resistance pattern of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae harbouring bla(CTX-M) in Glasgow, Scotland. METHODS: During a 12 week period, Enterobacteriaceae isolates obtained from urine samples were collected and susceptibility testing performed. Isolates were screened for the presence of bla(CTX-M) by multiplex PCR and selected Escherichia coli genes were subsequently sequenced. PFGE analysis was performed on selected E. coli isolates in order to identify clonal relationships. RESULTS: There were 155 phenotypically confirmed non-duplicate Enterobacteriaceae isolates obtained from urine samples. bla(CTX-M) was identified in 131/155 (84.5%) of the ESBL-producing isolates, with CTX-M group 1 enzymes accounting for 103/131 (78.6%) of these. The remaining 24 isolates carried other bla(CTX-M) types, including CTX-M group 2, CTX-M group 9 and an unidentifiable combination designated CTX-M group G2/Gx. A sample of 46/97 (47.4%) CTX-M-positive E. coli isolates was chosen for PFGE and demographic information regarding the source of the isolates was collated. Eight E. coli clusters were identified by PFGE; however, they did not achieve the 85% cut-off to demonstrate clonality. Nitrofurantoin resistance was significantly greater in the E. coli isolates expressing a non-CTX-M group 1 ESBL when compared with the E. coli isolates expressing a CTX-M group 1 ESBL. CONCLUSIONS: As seen in other British studies, bla(CTX-M) has become the predominant ESBL type in Glasgow, Scotland. The PFGE results show that four different CTX-M groups appear to be circulating in the community and within all four hospitals in the locality. There is little correlation between strain genotype and CTX-M group, thus it is unlikely that cross-infection alone is the driver. It is possible that plasmid migration of CTX-M genes within the E. coli population is occurring.

Epidemiology of Acinetobacter baumannii of animal origin.

Acinetobacter baumannii is an opportunistic pathogen responsible for nosocomial infections, however the origins of these bacteria remain unclear. Sixteen A. baumannii strains collected from animals slaughtered for human consumption were investigated for their susceptibility profiles, resistance islands (RIs), class 1 integrons, insertion sequence ISAba1, and bla(OXA-51)-like and bla(AmpC) genes. Polymerase chain reaction (PCR) and sequencing approaches were used to identify and type the isolates using the intrinsic gene bla(OXA-51)-like genes. Genotyping was also performed by pulsed-field gel electrophoresis (PFGE) to establish whether there was a genetic relationship between animal isolates and the main human isolates of European clones I, II and III (ECI, ECII and ECIII) known to cause major hospital outbreaks. All 16 isolates (100%) were sensitive to carbapenems, gentamicin, ciprofloxacin and piperacillin/tazobactam but were resistant to amoxicillin, cefradine, trimethoprim and chloramphenicol. Moreover, all isolates had a baseline resistance to ceftazidime, with a minimum inhibitory concentration of 4 mg/L. All isolates lacked RIs, ISAba1 and class 1 integrons but harboured bla(OXA-51)-like and bla(AmpC) genes. In addition, this study reports for the first time three new bla(OXA-51)-like genes (bla(OXA-148), bla(OXA-149) and bla(OXA-150)) isolated from bacteria in cattle, which have not been found previously in human isolates. However, all isolates recovered from pig faecal samples harboured one type of bla(OXA-51)-like (bla(OXA-51) itself), which has already been reported in human clinical isolates. From sequencing of the bla(OXA-51)-like genes from animal isolates, it was possible to identify four different clusters similar to those identified by PFGE, which in turn also distinguished these four groups from the human ECI, ECII and ECIII strains.