Detection of Extended-Spectrum ß-Lactamases among Acinetobacter Baumannii Isolated from Hospitals of Qazvin, Iran

BACKGROUND፡ Acinetobacter baumannii is a major contributor to nosocomial infections. Extended-spectrum ßlactamase (ESBL)-producing A. baumannii is spreading worldwide. We aimed to determine the frequency of ESBLencoding genes in clinical isolates of A. baumannii and to access their clonal relationship by repetitive extragenic palindromic-PCR (rep-PCR).METHODS: In this descriptive cross-sectional study, 203 isolates of A. baumannii were collected from Qazvin hospitals. The Identification of isolates was performed by standard laboratory methods. To verify ESBL production, all isolates were screened by disk agar diffusion and confirmed by the combined disk method. Subsequently, ESBL-encoding genes were detected by PCR and sequencing. Possible clonal association of ESBL-producing isolates was evaluated using rep-PCR.RESULTS: Two hundred (98.5%) isolates showed reduced susceptibility to one of the antibiotics used in the ESBL screening test, of which 127 isolates (62.6%) produced ESBL. PCR results showed blaOXA-1 (20.5%) was the most prevalent gene followed by blaTEM-1 (20%), blaGES-1 (15.7%), blaCTX-M-15 (7.9%), and blaPER-1(1.6%). Rep-PCR results revealed that ESBL-producing isolates belonged to clones A (85%), B (13.4%), and C (1.6%).CONCLUSION: Our study showed the significant presence of blaOXA-1, blaTEM-1, blaGES-1, blaCTX-M-15, and blaPER-1 genes in ESBLproducing A. baumannii isolates in the studied hospitals. This is the first report on the emergence of blaOXA-1 gene in these isolates in Iran. The use of comprehensive antimicrobial treatment guidelines based on laboratory data and appropriate infection control interventions are essential.

Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran

Acinetobacter baumannii, as a nosocomial pathogen has become a worldwide concern in recent years. In the current study, the resistance to tetracyclines and colistin were assessed in the isolates from different provinces of Iran.During the timeline of this study, a number of 270 isolates of A. baumannii were collected from tracheal aspirates, wounds, urine and blood cultures. The minimum inhibitory concentration (MIC) for tetracycline, doxycycline, minocycline, tigecycline and colistin were evaluated. Tetracycline resistance genes were assessed by PCR. The mean expression level of adeB, adeJ and adeG were assessed using semi quantitative Real-Time PCR. The clonal relationship of the isolates was evaluated by the repetitive extragenic palindromic PCR (REP-PCR), International Clonal (IC) Lineage Multiplex PCR and multilocus sequence typing (MLST) (Pasteur scheme) methods.The MIC by microdilution method showed that 87.5, 51.4, 28, 0.74 and 0% of the isolates were resistant to tetracycline, doxycycline, minocycline, tigecycline and colistin respectively. The prevalence of tetracycline resistance genes was 99.2, 99.2, 98, 86.7, 10, 3.33, 0.37, 0% for adeB, adeJ, adeG, tetB, tetA(39), tetA, tetM and tetH in tetracycline-resistant isolates. Moreover, the expression level of adeB, adeJ, adeG genes in tigecycline-nonsusceptible A. baumannii (TNAB) strain was higher compared to the tigecycline-susceptible A. baumannii (TSAB). A broad genomic diversity was revealed, but ST2 was the most prevalent ST. Our results indicated that tetracycline resistance in Iran is mediated by resistance-nodulation-cell division (RND) and tetB efflux pumps.

Multiple Drug-Resistant Vibrio Cholerae Responsible for Cholera Outbreak among Migrant Domestic Workers in Kerala, South India

Cholera outbreaks are prevalent in countries with a low Human Development Index (HDI) where people have limited access to safe drinking water, sanitation and hygiene (WASH). Intriguingly, the state of Kerala which records the highest HDI in India is endemic to cholera. We discuss the epidemiology of a cholera outbreak reported among migrant workers of Kerala in 2017. Virulence genes of Vibrio cholerae, toxR and ctxA, were detected in the river and ground water samples collected from the outbreaks sites which indicates need of enhanced awareness on WASH practices among migrant workers. The pathogenic Vibrio cholerae isolated from four patients in two districts had a similar DNA band pattern when analysed using repetitive extragenic palindromic-PCR (BOX), which indicates their single clonal origin. The four isolates were serotyped as O1 Ogawa. These isolates were resistant to multiple antibiotics including the carbapenem like imipenem. Nevertheless, isolates of these pathogenic bacteria were susceptible to tetracycline, identical to clinical isolates of Vibrio cholera reported earlier from this region. The current study highlights the importance of generating awareness on WASH protocols among migrant workers to prevent the outbreaks and considers community-based data as socioeconomic variable to predict the incidence of cholera.

Polyclonal gut colonization with extended-spectrum cephalosporin- and/or colistin-resistant Enterobacteriaceae: a normal status for hotel employees on the island of Zanzibar, Tanzania

Objectives For low-income countries, data regarding the intestinal colonization with extended-spectrum cephalosporin-resistant (ESC-R) and colistin-resistant (CST-R) Enterobacteriaceae in the community are still scarce. Here, we investigated this phenomenon by analysing hotel employees in Zanzibar. Methods During June to July 2018, rectal swabs from 59 volunteers were screened implementing selective enrichments and agar plates. Species identification was achieved using MALDI-TOF MS. Strains were characterized using microdilution panels (MICs), microarray, PCRs for mcr-1/-8, repetitive extragenic palindromic-PCR (rep-PCR) and WGS. Results Colonization prevalence with ESC-R-, CST-R- and mcr-1-positive Enterobacteriaceae were 91.5%, 66.1% and 18.6%, respectively (average: 2.2 strains per volunteer). Overall, 55 ESC-R Escherichia coli (3 also CST-R), 33 ESC-R Klebsiella pneumoniae (1 also CST-R), 17 CST-R E. coli and 21 CST-R K. pneumoniae were collected. The following main resistance genes were found: ESC-R E. coli (blaCTX-M-15-like, 51.0%), ESC-R K. pneumoniae (blaCTX-M-9-like, 42.9%), CST-R E. coli (mcr-1, 55%) and CST-R K. pneumoniae (D150G substitution in PhoQ). ESBL-producing E. coli mainly belonged to ST361, ST636 and ST131, whereas all those that were mcr-1 positive belonged to ST46 that carried mcr-1 in a 33 kb IncX4 plasmid. ESBL-producing K. pneumoniae mainly belonged to ST17, ST1741 and ST101, whereas CST-R strains belonged to ST11. Conclusions We recorded remarkably high colonization prevalence with ESC-R and/or CST-R Enterobacteriaceae in hotel staff. Further research in the local environment, livestock and food chain is warranted to understand this phenomenon. Moreover, as Zanzibar is a frequent holiday destination, attention should be paid to the risk of international travellers becoming colonized and thereby importing life-threatening pathogens into their low-prevalence countries.

Genetic Relatedness of Salmonella Serovars Isolated from Catfish (Clarias gariepinus) and Tilapia (Tilapia mossambica) Obtained from Wet Markets and Ponds in Penang, Malaysia

ABSTRACT A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovis-morbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.

In VivoEvolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli Sequence Type 131: Characterization of an Acquired Extended-Spectrum AmpC Conferring Resistance to Cefepime

ABSTRACTCefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identicalEscherichia coliisolates obtained from a patient. The CMY-2-producingE. coliisolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producingE. coliisolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ecand CMY-2-Ecwere identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤0.5 μg/ml for CMY-33-Ecand CMY-2-Ec, respectively). Identical CMY-2-Ecisolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed inE. colistrain DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). Thekcat/Kmor inhibitor complex inactivation (kinact)/Kiapp(μM−1s−1) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the firstin vivodemonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ecisolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.

Prevalence and Genetic Characterization of Vibrio vulnificus in Raw Seafood and Seawater in Malaysia

Vibrio vulnificus is a highly invasive human pathogen that exists naturally in estuarine environment and coastal waters. In this study, we used different PCR assays to detect V. vulnificus in 260 seafood and 80 seawater samples. V. vulnificus was present in about 34 (13%) of the 260 seafood samples and 18 (23%) of the 80 seawater samples. Repetitive extragenic palindromic PCR (REP-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) were applied to subtype the V. vulnificus isolates. Twenty-five REP profiles and 45 ERIC profiles were observed, and the isolates were categorized into 9 and 10 distinct clusters at the similarity of 80%, by REP-PCR and ERIC-PCR, respectively. ERIC-PCR is more discriminative than REP-PCR in subtyping V. vulnificus, demonstrating high genetic diversity among the isolates.

Emergence of Extensively Drug-Resistant Haemophilus parainfluenzae in Switzerland

ABSTRACTTwo homosexual men were colonized in the urethra withHaemophilus parainfluenzaenonsusceptible to ampicillin (MIC, 8 μg/ml), amoxicillin-clavulanate (MIC, 4 μg/ml), cefotaxime (MIC, 1.5 μg/ml), cefepime (MIC, 3 μg/ml), meropenem (MIC, 0.5 μg/ml), cefuroxime, azithromycin, ciprofloxacin, tetracycline, and chloramphenicol (all MICs, ≥32 μg/ml). Repetitive extragenic palindromic PCR (rep-PCR) showed that the strains were indistinguishable. The isolates had amino acid substitutions in PBP3, L4, GyrA, and ParC and possessed Mef(A), Tet(M), and CatS resistance mechanisms. This is the first report of extensively drug-resistant (XDR)H. parainfluenzae.