Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla _TEM-1 and bla _CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.     

Selection of broad‐spectrum cephalosporin‐resistant Escherichia coli in the feces of healthy dogs after administration of first‐generation cephalosporins

Although antimicrobial products are essential for treating diseases caused by bacteria, antimicrobial treatment selects for antimicrobial‐resistant (AMR) bacteria. The aim of this study was to determine the effects of administration of first‐generation cephalosporins on development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)‐resistant E. coli in fecal samples of three healthy dogs treated i.v. with cefazolin before castration and then orally with LEX for 3 days post‐operation (PO) were examined using DHL agar with or without LEX (50 µg/mL). LEX‐resistant E. coli were found within 3 days PO, accounted for 100% of all identified E. coli 3–5 days PO in all dogs, and were predominantly found until 12 days PO. LEX‐resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed‐field gel electrophoresis (PFGE) genotyping, β‐lactamase typing and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration ≥4 µg/mL). Seven PFGE profiles were classified into five groups and three β‐lactamase combinations (bla_CMY‐4‐bla_TEM‐1, bla_TEM‐1‐bla_CTX‐M‐15 and bla_TEM‐1‐bla_CTX‐M‐15‐bla_CMY‐4). All isolates exhibited identical PFGE profiles in all dogs on four days PO and subsequently showed divergent PFGE profiles. Our results indicate there are two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuing hygiene practices are necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.     

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers,pigs and meat products in Thailand and Cambodia provinces

Nine hundred and forty‐one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36–85% in Sa Keao and 11–69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1‐aadA1, dfrA12‐aadA2 and aadA2‐linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12‐aadA2) and one broiler isolate from Banteay Meanchey (dfrA1‐aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL‐producing strains with ESBL gene bla_CTX‐M‐15, broad spectrum β‐lactamase gene bla_TEM‐1 and the AmpC gene bla_CMY‐2 being detected. The bla_TEM‐1 gene was most prevalent and located on a conjugative plasmid.     

Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil

The aim of the present study was to investigate the resistance profile, to detect the presence of beta-lactam resistance genes, phenotypic expression of efflux pump systems and class 1 integrons in Pseudomonas spp. strains obtained from untreated hospital effluents. Effluent samples were collected from four hospitals in Porto Alegre, RS, Brazil. Pseudomonas were isolated on MacConkey agar plates and the identification was confirmed by 16S rRNA PCR and biochemical tests. Susceptibility testing was determined by disk-diffusion method using 11 different beta-lactams and MIC assays were performed on isolates resistant to imipenem and ceftazidime. The beta-lactamase genes bla _IMP, bla _VIM, bla _SPM-1, bla _OXA-23-like, bla _OXA-24-like, bla _OXA-51-like and the intl1 gene from class 1 integron were analysed by PCR. One hundred and twenty-four isolates were recovered and the most common species was Pseudomonas pseudoalcaligenes. The resistance found among the isolates was considered high, 62 (50%) isolates were multiresistant. No isolate carrying the beta-lactamase genes tested was found among the strains. Seven isolates showed reduction of MIC for imipenem and ceftazidime in the presence of cyanide m-chlorophenylhydrazone, indicating the hyper expression of efflux pumps. From the 124 isolates, 52 (41.9%) were identified as carrying the class 1 integron gene, intI1. Untreated hospital effluents could be a source of environmental contamination due to discharge of antimicrobial resistant bacteria which can carry integron class 1 and act as a reservoir of resistance genes and have efflux pump systems.     

Multiresistance, beta-lactamase-encoding genes and bacterial diversity in hospital wastewater in Rio de Janeiro, Brazil

Aims: To investigate the bacterial diversity, antimicrobial resistance patterns and types of beta‐lactamase genes in Gram‐negative bacteria isolated from a hospital sewage treatment plant. Methods and Results: Between July and December 2008, we collected samples from influent, clarifier tank effluent and chlorine contact tank effluent from a sewage treatment plant service of a hospital located in the city of Rio de Janeiro, Brazil. Of the 221 isolates identified, 40% were characterized as extended‐spectrum beta‐lactamase (ESBL) producers. Nonpathogenic micro‐organisms and some pathogenic genera were quantified. The most common ESBL‐producing isolates were Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli. The bla_TEM, bla_SHV and bla_CTX‐M genes were detected in 82, 48 and 67% of bacterial isolates, respectively. Conclusions: Results showed that hospital wastewater treatment plant is not suitable systems for the removal of all antibiotic‐resistant micro‐organisms present in hospital wastewaters. Significance and Impact of the Study: This study provides evidence that bacteria resistant to multiple antibiotics and their resistance genes that are usually present in the hospital can reach the environment, even after the use of hospital wastewater treatment plants.     

Prevalence and antimicrobial resistance in Salmonella enterica isolated from broiler chickens,pigs and meat products in Thailand–Cambodia border provinces

This study aimed to examine the prevalence and antimicrobial resistance (AMR) of Salmonella isolates from broiler chickens, pigs and their associated meat products in the Thailand–Cambodia border provinces. A total of 941 samples were collected from pigs and broiler chickens at slaughter houses and from carcasses at local fresh markets in Sa Kaeo, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) in 2014 and 2015. From these samples, 345 Salmonella isolates were collected from Sa Keao (n = 145; 23%) and Banteay Meanchey (n = 200; 47%) and assayed for antimicrobial susceptibility, class 1 integrons and extended‐spectrum β‐lactamase (ESBL) genes. Serovars Typhimurium (29%) and Rissen (29%) were the most common serotypes found in Thai and Cambodian isolates, respectively. Multidrug resistance was detected in 34% and 52% of isolates from Sa Keao and Banteay Meanchey, respectively. The majority of the Thai isolates were resistant to ampicillin (72.4%), whereas most Cambodian isolates were resistant to sulfamethoxazole (71%). Eleven isolates from Sa Keao and 44 from Banteay Meanchey carried class 1 integrons comprising resistance gene cassettes. The most common gene cassette array was dfrA12‐aadA2 (61.1%). Six isolates were ESBL producers. The β‐lactamase genes found included bla_TEM‐1, bla_CTX‐M‐55 and bla_CMY‐2. Some of these class 1 integrons and ESBL genes were located on conjugative plasmid. In conclusion, multidrug‐resistant Salmonella are common in pigs, chickens and their products in the Thailand–Cambodia border provinces. Our findings indicate that class 1 integrons play a role in spread of AMR in the strains in this study.     

Incidence of diverse integrons and β-lactamase genes in environmental <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> isolates from Jiaozhou Bay,China

Environmental microbiology investigation was performed to determine the molecular diversity of β-lactamase genes among ampicillin-resistant bacteria from Jiaozhou Bay. β-lactamase genes were detected in 93.8% of the bacterial isolates identified as Enterobacteriaceae. The most frequently detected gene was bla _TEM, followed by bla _SHV, bla _OAX-1, bla _MOX and bla _CMY. Most of the isolates (68.8%) were positive for the intI1 integrase gene, and two isolates were also found for the intI2 gene. The dfr and aadA gene cassettes were predominant. Anthropogenic contamination from onshore sewage processing plants might contribute predominantly to the β-lactamase gene reservoir in the studied coastal waters. Environmental antibiotic-resistant bacteria and resistance genes may serve as bioindicators of coastal environmental quality or biotracers of the potential contamination sources. This is the first report of the prevalence and characterization of β-lactamase genes and integrons in coastal Enterobacteriaceae from China.     

Influence of agricultural practice on mobile bla genes: IncI1‐bearing CTX‐M,SHV, CMY and TEM in Escherichia coli from intensive farming soils

Many calls have been made to address antibiotic resistance in an environmental perspective. With this study, we showed the widespread presence of high‐level antibiotic resistant isolates on a collection of non‐susceptible Gram‐negative bacteria (n = 232) recovered from soils. Bacteria were selected using amoxicillin, cefotaxime and imipenem, from sites representing different agricultural practices (extensive, intensive and organic). Striking levels of non‐susceptibility were noticed in intensive soils for norfloxacin (74%), streptomycin (50.7%) and tetracycline (46.6%); indeed, the exposure to intensive agricultural practices constituted a risk factor for non‐susceptibility to many antibiotics, multidrug resistance and production of extended‐spectrum β‐lactamases (ESBL). Analyses of non‐susceptibility highlighted that environmental and clinical bacteria from the same species might not share the same intrinsic resistance patterns, raising concerns for therapy choices in environment‐borne infections. The multiple sequence‐type IncI1‐driven spread of penicillinases (bla_TEM‐1, bla_TEM‐135), ESBL (bla_SHV‐12 and bla_CTX‐M‐1) and plasmid‐mediated AmpC β‐lactamases (bla_CMY‐2), produced by isolates that share their molecular features with isolates from humans and animals, suggests contamination of agricultural soils. This is also the first appearance of IncI1/ST28‐harbouring bla_CTX‐M‐1, which should be monitored to prevent their establishment as successfully dispersed plasmids. This research may help disclose paths of contamination by mobile antibiotic resistance determinants and the risks for their dissemination.     

Occurrence of the genes encoding carbapenemases,ESBLs and class 1 integron-integrase among fermenting and non-fermenting bacteria from retail goat meat

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n = 57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using bla_NDM-positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20 kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the bla_KPC, bla_NDM, bla_SHV and bla_TEM genes, respectively. The bla_KPC-2 gene was observed in one E. coli isolate. The bla_NDM-1 gene was detected in Stenotrophomonas maltophilia (n = 2), Acinetobacter baumannii (n = 1) and Ochrobactrum anthropi (n = 1) isolates. These isolates co-harboured the bla_TEM and bla_SHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of bla_NDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks.     

Detection of blaKPC and blaNDM genes by duplex PCR with lateral flow dipsticks from sterile body fluid samples

Duplex polymerase chain reaction with lateral flow dipsticks (duplex PCR-LFD) was developed for the simultaneous detection of beta-lactamase Klebsiella pneumoniae carbapenemase (bla_KPC) and beta-lactamase New Dehli metallo-beta-lactamase (bla_NDM) genes in body fluid samples. This method was validated using well-characterized isolates. The assessment of the specificity of duplex PCR-LFD showed that there was no cross-reactivity with other targets. The detection limit of the duplex PCR-LFD assay was 20 CFU per ml for bla_KPC and bla_NDM. Among 177 sterile body fluid samples tested by the duplex PCR-LFD assay, 40 were bla_KPC-positive and five were bla_NDM-positive. The results obtained from 122 corresponding Gram-negative bacteria which were isolated from these clinical samples and tested by duplex PCR-LFD assay showed that there were 37 strains carrying bla_KPC genes in 40 bla_KPC-positive samples and three strains carrying bla_NDM genes in five bla_NDM-positive samples. Statistical analysis indicated that there was no significant difference between the direct detection of bla_KPC and bla_NDM genes in clinical sterile body fluid samples and their corresponding clinical isolates. Therefore, duplex PCR-LFD can be effective for the simultaneous detection of bla_KPC and bla_NDM in clinical isolates and directly from clinical samples, which may be helpful for the administration of appropriate antimicrobial treatment.     

Prevalence of the <Emphasis Type="Italic">bla</Emphasis><Subscript>SHV</Subscript> Gene in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Isolates Obtained from Hospital and Community Infections and from the Microbiota of Healthy Individuals in Recife,Brazil

The aim of this study was to determine the prevalence of the bla _SHV gene in Klebsiella pneumoniae isolates from hospital and community infections and from the normal microbiota of healthy individuals in Recife, PE, Brazil. Fifty-two K. pneumoniae isolates were analyzed regarding the presence of the bla _SHV gene, using PCR, and eight isolates were analyzed by DNA sequencing. This gene was detected in 16 isolates from hospital infections, four from community infections, and nine from the normal microbiota. This was the first study to find the bla _SHV gene in K. pneumoniae isolates from the normal microbiota. Through DNA sequencing of eight K. pneumoniae isolates from hospital and community infections, with a resistance phenotype indicative of extended-spectrum β-lactamase production, a new SHV variant named SHV-122 was found. We also detected the presence of bla _SHV-1, bla _SHV-11, bla _SHV-28, and bla _SHV-108. The results show that in Recife, Brazil, K. pneumoniae isolates that presented resistance to oxyimino-β-lactams had high prevalence and diversity of the bla _SHV gene. We also conclude that there was a high presence of the bla _SHV gene among isolates from the normal microbiota of healthy individuals.     

污水厂产超广谱β内酰胺酶大肠杆菌通过接合水平传递耐药性

【目的】研究废水中产超广谱β-内酰胺酶大肠杆菌中可移动质粒在耐药基因水平传播机制中的作用。【方法】对污水厂分离所得的50株产ESBLs大肠杆菌进行接合试验,并对所得的接合子采用纸片扩散法测定其对15种常见药物的耐药表型,针对质粒介导的产ESBLs菌株的耐药基因设计7对特异性引物对接合子进行PCR扩增。【结果】研究结果显示,80份水样分离得50株产ESBLs大肠杆菌,共接合成功35株细菌,接合成功率高达70%。接合子与供体菌相比,均发生耐药谱型的改变,且存在丢失一种或几种药物耐药性且产生另一种或几种药物耐药性的现象。PCR扩增结果显示,接合子与供体菌相比,耐药基因型有所减少或不变,bla_(TEM)、bla_(CTX-M)基因全部接合成功,bla_(SHV)基因仅1株未接合成功,耐氟喹诺酮类基因未发生转移。【结论】本研究表明,不同的耐药基因可能位于不同的可移动质粒上,可移动质粒在大肠杆菌耐药性水平传播的过程中起到了十分重要的作用。     

Analysis of a novel class 1 integron containing metallo-β-lactamase gene VIM-2 in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Carbapenems such as imipenem are stable to most β-lactamases. Recently, increased numbers of carbapenemase producing Gram-negative bacterial strains have been isolated because of the increased use of cabapenems. In this respect, control of these infectious carbapenemase producing Gram-negative bacteria and understanding their resistance mechanism are becoming more important. These carbapenem-hydrolyzing β-lactamase genes have been reported to exist mostly as gene cassettes in an integron. This implies that antibiotic resistance genes may be transferred to other bacteria via the integron. In the present study, we identified and analyzed an integron containing VIM-2 type metallo-β-lactamase gene in a carbapenemase producing Pseudomonas aeruginosa. In addition, the possibility of resistance spread by integron located in a plasmid was tested. Among glucose non-fermenting Gram-negative bacilli with reduced imipenem susceptibility (MIC≥8 μg/ml) isolated from Korean patients, P. aeruginosa 1082 showed resistance to most β-lactams, cephalosporin, and aminoglycoside. We found that P. aeruginosa 1082 was inhibited by EDTA in EDTA double disk synergy test which means that this strain produces metallo-β-lactamase. Class 1 integron containing bla _VIM-2 (carbapenem resistance gene), qacF (quaternary ammonium compound resistance gene), aacA4 (aminoglycoside resistance gene), catB3 (chloramphenicol resistance gene), bla _oxa-30 (extended-spectrum β-lactam resistance gene), and aadAl (aminoglycoside resistance gene) gene cassettes was detected in P. aeruginosa 1082. The size of the integron was 5,246 bp and the structure and arrangement of the integron was a novel one in comparison with other integrons found in other P. aeruginosa. The integron could be transferred to Escherichia coli JM109 from P. aeruginosa 1082 possibly via self-transferable plasmid DNA. The integron and a bla _VIM-2 gene were detected in the plasmid DNA of the transconjugants whose imipenem resistance was slightly increased as a result of accepting the integron from the donor strain.     

Carbapenem-Resistant Bacteria Recovered from Faeces of Dairy Cattle in the High Plains Region of the USA

Objective

A study was conducted to recover carbapenem-resistant bacteria from the faeces of dairy cattle and identify the underlying genetic mechanisms associated with reduced phenotypic susceptibility to carbapenems.

Methods

One hundred and fifty-nine faecal samples from dairy cattle were screened for carbapenem-resistant bacteria. Phenotypic screening was conducted on two media containing ertapenem. The isolates from the screening step were characterised via disk diffusion, Modified Hodge, and Carba NP assays. Carbapenem-resistant bacteria and carbapenemase-producing isolates were subjected to Gram staining and biochemical testing to include Gram-negative bacilli. Whole genome sequencing was performed on bacteria that exhibited either a carbapenemase-producing phenotype or were not susceptible to ertapenem and were presumptively Enterobacteriaceae.

Results

Of 323 isolates collected from the screening media, 28 were selected for WGS; 21 of which were based on a carbapenemase-producing phenotype and 7 were presumptively Enterobacteriaceae and not susceptible to ertapenem. Based on analysis of WGS data, isolates included: 3 Escherichia coli harbouring bla_CMY-2 and truncated ompF genes; 8 Aeromonas harbouring bla_cphA-like genes; 1 Acinetobacter baumannii harbouring a novel bla_OXA gene (bla_OXA-497); and 6 Pseudomonas with conserved domains of various carbapenemase-producing genes.

Conclusions

Carbapenem resistant bacteria appear to be rare in cattle. Nonetheless, carbapenem-resistant bacteria were detected across various genera and were found to harbour a variety of mechanisms conferring reduced susceptibility. The development and dissemination of carbapenem-resistant bacteria in livestock would have grave implications for therapeutic treatment options in human medicine; thus, continued monitoring of carbapenem susceptibility among enteric bacteria of livestock is warranted.     

Dissemination of ceftazidime-resistant Acinetobacter baumannii clonal complex 92 in Korea

Aims: This study was performed to describe the epidemiological traits of ceftazidime‐resistant Acinetobacter baumannii clinical isolates from Korea. Methods and Results: Antimicrobial susceptibilities were determined by disk diffusion assay. PCR experiments were performed to detect genes encoding extended‐spectrum β‐lactamases and metallo‐β‐lactamases. Detection of ISAba1 upstream of the bla_ADC gene was also performed by PCR amplification. The genetic organization of the bla_PER‐1 gene was investigated by PCR mapping and sequencing of the regions surrounding the gene. Multilocus sequence typing was performed using seven housekeeping genes. A. baumannii isolates of clonal complex (CC) 92 exhibited a higher resistance rate (286/289, 99%) against ceftazidime compared to A. baumannii isolates of non‐CC92 (7/87, 8%). Amongst 286 ceftazidime‐resistant isolates of CC92, 100 (35%) isolates carried the bla_PER‐1 gene, while none of the 87 isolates of non‐CC92 carried the gene. The bla_ADC gene associated with an ISAba1 element was detected in 98% (281/286) of ceftazidime‐resistant isolates of CC92 and in all seven ceftazidime‐resistant isolates of non‐CC92. The bla_PER‐1 gene was located on a transposon, Tn1213 (ISPa12‐bla_PER‐1‐Δgst‐ISPa13), in 95 isolates and on a complex class 1 integron (orf513‐bla_PER‐1‐putative ABC transporter gene) in five isolates. Southern blot experiments confirmed the chromosomal location of the bla_PER‐1 gene. Conclusions: Acinetobacter baumannii CC92 which has acquired ceftazidime resistance by the production of PER‐1 extended‐spectrum β‐lactamases and/or the overproduction of Acinetobacter‐derived cephalosporinase is widely disseminated in Korea. Significance and Impact of the Study: This study shows the mechanisms of acquiring ceftazidime resistance in A. baumannii and the epidemiological traits of ceftazidime‐resistant A. baumannii isolates from Korea.     

克雷伯氏菌属(Klebsiella)细菌比较基因组学分析揭示多复制子抗性质粒介导广泛的抗性基因传播

[目的] 研究克雷伯氏菌与多复制子抗性质粒间的关系,分析细菌携带多复制子质粒对抗生素环境的响应机制。[方法] 以2018-2020年分离的56株不同来源克雷伯氏菌（Klebsiella sp.）分离株为研究对象,利用微量肉汤稀释法评估其多重耐药表型,对分离菌株进行全基因组测序（WGS）,通过细菌全基因组关联分析（BGWAS）技术和比较基因组学方法深入解析多复制子抗性质粒形成的机制。[结果] 耐药表型分析发现野生动物来源的菌株具有更广的耐药谱系,总体Klebsiella sp.对氨苄西林表现出很高的耐药率（80.36%）,尤其是马来穿山甲来源菌株对头孢类抗生素高度耐受,同时对氯霉素、左氧氟沙星和复方新诺明等药物耐受,基因组分析发现这些菌株携带了抗性质粒和更多的抗生素抗性基因。进一步对69个质粒序列分析,发现有28个质粒为多复制子质粒,主要携带bla_CTX-M-15、bla_CTX-M-14、bla_CTX-M-55、bla_OXA-1和bla_TEM-1等β-内酰胺酶基因。细菌携带质粒类型分析认为Klebsiella pneumoniae可能是多复制子质粒的重要宿主,质粒骨架与结构分析发现多复制子质粒多由2个或2个以上单个质粒融合而成,携带此类质粒的菌株不仅获得了更广的耐药表型,而且在全球传播扩散分布逐年增加,因此产生对抗生素环境更强的适应性。[结论] 多重耐药性细菌呈现的表型与携带的多复制子质粒有关,相比较下多复制子质粒比非多复制子质粒有更强的抗性基因携带能力,或许是细菌在强大的抗生素压力下产生的重要响应机制。本研究对于未来探索细菌抗性基因的传播扩散机制具有重要意义。     

Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital

Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla _CTX-M-15 (12 isolates), bla _CTX-M-14a (one isolate), and bla _CTX-M-14b (one isolate). The bla _OXA-1 gene was detected in 13 bla _CTX-M-producing strains and a bla _TEM-1 gene in 6 of them. The ISEcp1 sequence was found upstream of bla _CTX-M genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17–aadA5 being the most common. One of the strains (bla _CTX-M-14a-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6′)-Ib and cmlA1 genes and it was linked to the bla _CTX-M-14a gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.     

Co-occurrence of genes encoding carbapenemase,ESBL, pAmpC and non-β-Lactam resistance among Klebsiella pneumonia and E. coli clinical isolates in Tunisia

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried bla_NDM-1 (n = 11), bla_OXA-48 (n = 11), bla_NDM-1 + bla_OXA-48 (n = 1), bla_NDM-1 + bla_VIM-1 (n = 1), bla_OXA-204 (n = 1), along with bla_CTX-M, bla_OXA, bla_TEM, bla_CMY, bla_DHA and bla_SHV genes variants on conjugative plasmid of IncL/M, IncR, IncFII_K, IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.     

Molecular characteristics and resistant mechanisms of imipenem-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> isolates in Shenyang,China

The investigation was carried out to elucidate the molecular characteristics and resistant mechanisms of imipenem-resistant Acinetobacter baumannii. Thirty-seven isolates were collected from January 2007 to December 2007. The homology of the isolates was analyzed by both pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genes of β-lactamases, adeB, and class 1 integron were polymerase chain reaction amplified. Genotype analysis of the 37 A. baumannii isolates by PFGE revealed the circulation of four PFGE types (A-D); the A- and B-type accounted for 48.6% and 40.5%, respectively. MLST showed the existence of three allelic profiles. The agar dilution method was carried out to determine the MIC of imipenem, in the absence or presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μg/ml). The MICs of the strains to imipenem were between 16 μg/ml and 128 μg/ml. When CCCP was added, a MIC decrease of at least four-fold was observed in 20 isolates, which belonged to the A- or C-type. AdeB and bla _PER-1 genes were each detected in 35 isolates, bla _OXA-23 gene in 34 isolates and bla _OXA-58-like gene in 24 isolates. All isolates harbored bla _OXA-51-like genes. No isolates carried the bla _IMP-1 gene. Integron was detected in 25 isolates, which mediated the resistance to aminoglycosides and rifampin. The epidemiologic data suggested that the increasing infection of A. baumannii in our hospital was mainly caused by the inter-hospital spread of two epidemic clones. The AdeABC efflux system may be the important factor that leads to the high level of imipenem-resistance in PFGE A-type.     

Study of CTX-M Type of Extended Spectrum β-Lactamase among Nosocomial Isolates of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in South India

Data on CTX-M type extended-spectrum β-lactamase (ESBL) produced by Gram-negative bacteria by molecular methods are limited from India. This study was conducted to investigate the prevalence of CTX-M type ESBL producing Escherichia coli and Klebsiella pneumoniae from nosocomial isolates in a tertiary care hospital in southern India. A total of 179 clinical isolates of K. pneumoniae (n = 72) and E. coli (n = 107) were obtained in a period of 3 months and assessed for ESBL production phenotypically. Associated resistance to a panel of antibiotics and Minimum Inhibitory Concentration for 3rd generation cephalosporins was determined. Phenotypically ESBL positive isolates were subjected to PCR for bla _CTX-M gene using two sets of primers for the simultaneous detection of all the five major groups of CTX-M types. All the positive isolates were then subjected to a group specific PCR to detect the prevalent group. Out of 179 isolates, 156 (87.1%) were positive for ESBL phenotypically, which includes 39.2% of K. pneumoniae and 60.8% of E. coli. All of them were examined by PCR using two primers for the presence of bla _CTX-M genes. Among the 156 phenotypic positive isolates, 124 (79.4%) were positive for bla _CTX-M genes, of which 45 (36.2%) were K. pneumoniae, 79 (63.7%) were E. coli. When the 124 positive clinical isolates were further tested with CTX-M group-specific primers, all were positive for the CTX-M-1 group. Our findings document evidence of the high prevalence of multidrug resistant CTX-M group 1 type ESBL among nosocomial isolates in this region. High co-resistance to other non-β-lactam antibiotics is a major challenge for management of ESBL infections. This is alarming and calls for the judicious use of carbapenems, especially in developing countries. This has significant implications for patient management, and indicates the need for increased surveillance and for further molecular characterization of these isolates.