Plasmid-mediated Antimicrobial Resistance Genes Research Articles

Carbapenem-Resistant Enterobacter cloacae Complex in Southwest China: Molecular Characteristics and Risk Factors Caused by NDM Producers.

The isolation rate of carbapenem-resistant Enterobacter cloacae complex (CREC) is continuously increasing. The aims of this study were to investigate the molecular characteristics and risk factors associated with CREC infections. Bacterial species were identified using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany), and the hsp60 gene was utilized for further typing. Antimicrobial susceptibilities were assessed through the MicroScan WalkAway 96 Plus system (Siemens, Germany) and the microbroth dilution method. Antimicrobial resistance genes were screened through polymerase chain reaction (PCR), while the homologous relationship was assessed using multilocus sequence typing (MLST). Conjugation experiments were performed to verify whether the plasmid could be transferred. Additionally, logistic regression model was employed to analyze risk factors for CREC infections. 32 strains of CREC bacteria were isolated during the study, yet only 20 were retained for preservation. While the isolates demonstrated resistance to the majority of antibiotics, they exhibited high sensitivity to polymyxin B and tigecycline. All isolates carried the blaNDM resistance gene, including 13 blaNDM-1 isolates and 7 blaNDM-5 isolates. MLST homology analysis revealed the presence of seven known ST types and one new ST type. Conjugation experiments confirmed that 13 isolates were capable of transferring the blaNDM resistance gene to Escherichia coli strain EC600. Single-factor analysis identified multiple primary risk factors for CREC infection, but multivariate analysis did not reveal independent risk factors. This study investigates the molecular characteristics and risk factors associated with CREC infections. The detection rate of CREC strains in our hospital is continuously rising and homology analysis suggested that strains might spread in our hospital, emphasizing the importance of implementing effective preventive measures to control the horizontal transmission of plasmid-mediated antimicrobial resistance genes.

Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs.

Monitoring the prevalence of antimicrobial resistance genes (ARGs) is vital for addressing the global crisis of antibiotic-resistant bacterial infections. Despite its importance, the characterization of ARGs and microbiome structures, as well as the identification of indicators for routine ARG monitoring in pig farms, are still lacking, particularly concerning variations in antimicrobial exposure in different countries or regions. Here, metagenomics and random forest machine learning were used to elucidate the ARG profiles, microbiome structures, and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe. Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs ( P<0.05). ANT(6)-Ib, APH(3')-IIIa, and tet(40) were identified as shared core ARGs between the two pig populations. Furthermore, the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions. Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs, respectively. Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100% and 98.7%, respectively. Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy ( r=0.72-0.88). Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs. The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

Metagenomic Insight into Microbiome and Antibiotic Resistance Genes of High Clinical Concern in Urban and Rural Hospital Wastewater of Northern India Origin: a Major Reservoir of Antimicrobial Resistance.

India is one of the largest consumers and producers of antibiotics and a hot spot for the emergence and proliferation of antimicrobial resistance genes (ARGs). Indian hospital wastewater (HWW) accumulates ARGs from source hospitals and often merges with urban wastewater, with the potential for environmental and human contamination. Despite its putative clinical importance, there is a lack of high-resolution resistome profiling of Indian hospital wastewater, with most studies either relying on conventional PCR-biased techniques or being limited to one city. In this study, we comprehensively analyzed antibiotic resistomes of wastewater from six Indian hospitals distributed in rural and urban areas of northern India through shotgun metagenomics. Our study revealed the predominance of ARGs against aminoglycoside, macrolide, carbapenem, trimethoprim, and sulfonamide antibiotics in all the samples through both read-based analysis and assembly-based analysis. We detected the mobile colistin resistance gene mcr-5.1 for the first time in Indian hospital sewage. blaNDM-1 was present in 4 out of 6 samples and was carried by Pseudomonas aeruginosa in HWW-2, Klebsiella pneumoniae in HWW-4 and HWW-6, and Acinetobacter baumanii in HWW-5. Most ARGs were plasmid-mediated and hosted by Proteobacteria. We identified virulence factors and transposable elements flanking the ARGs, highlighting the role of horizontal gene transmission of ARGs. IMPORTANCE There is a paucity of research on detailed antibiotic resistome and microbiome diversity of Indian hospital wastewater. This study reports the predominance of clinically concerning ARGs such as the beta-lactamases blaNDM and blaOXA and the colistin resistance gene mcr and their association with the microbiome in six different Indian hospital wastewaters of both urban and rural origin. The abundance of plasmid-mediated ARGs and virulence factors calls for urgent AMR crisis management. The lack of proper wastewater management strategies meeting international standards and open drainage systems further complicates the problem of containing the ARGs at these hospitals. This metagenomic study presents the current AMR profile propagating in hospital settings in India and can be used as a reference for future surveillance and risk management of ARGs in Indian hospitals.

Understanding the Mechanism of Antimicrobial Resistance and Pathogenesis of Salmonella enterica Serovar Typhi.

Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative pathogen that causes typhoid fever in humans. Though many serotypes of Salmonella spp. are capable of causing disease in both humans and animals alike, S. Typhi and S. Paratyphi are common in human hosts only. The global burden of typhoid fever is attributable to more than 27 million cases each year and approximately 200,000 deaths worldwide, with many regions such as Africa, South and Southeast Asia being the most affected in the world. The pathogen is able to cause disease in hosts by evading defense systems, adhesion to epithelial cells, and survival in host cells in the presence of several virulence factors, mediated by virulence plasmids and genes clustered in distinct regions known as Salmonella pathogenicity islands (SPIs). These factors, coupled with plasmid-mediated antimicrobial resistance genes, enable the bacterium to become resistant to various broad-spectrum antibiotics used in the treatment of typhoid fever and other infections caused by Salmonella spp. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains in many countries of the world has raised great concern over the rise of antibiotic resistance in pathogens such as S. Typhi. In order to identify the key virulence factors involved in S. Typhi pathogenesis and infection, this review delves into various mechanisms of virulence, pathogenicity, and antimicrobial resistance to reinforce efficacious disease management.

Genomic characterization of multidrug-resistant Salmonella serovar Kentucky ST198 isolated in poultry flocks in Spain (2011-2017).

Salmonella Kentucky is commonly found in poultry and rarely associated with human disease. However, a multidrug-resistant (MDR) S. Kentucky clone [sequence type (ST)198] has been increasingly reported globally in humans and animals. Our aim here was to assess if the recently reported increase of S. Kentucky in poultry in Spain was associated with the ST198 clone and to characterize this MDR clone and its distribution in Spain. Sixty-six isolates retrieved from turkey, laying hen and broiler in 2011–2017 were subjected to whole-genome sequencing to assess their sequence type, genetic relatedness, and presence of antimicrobial resistance genes (ARGs), plasmid replicons and virulence factors. Thirteen strains were further analysed using long-read sequencing technologies to characterize the genetic background associated with ARGs. All isolates belonged to the ST198 clone and were grouped in three clades associated with the presence of a specific point mutation in the gyrA gene, their geographical origin and isolation year. All strains carried between one and 16 ARGs whose presence correlated with the resistance phenotype to between two and eight antimicrobials. The ARGs were located in the Salmonella genomic island (SGI-1) and in some cases (bla SHV-12 , catA1, cmlA1, dfrA and multiple aminoglycoside-resistance genes) in IncHI2/IncI1 plasmids, some of which were consistently detected in different years/farms in certain regions, suggesting they could persist over time. Our results indicate that the MDR S. Kentucky ST198 is present in all investigated poultry hosts in Spain, and that certain strains also carry additional plasmid-mediated ARGs, thus increasing its potential public health significance.

Public health implications of plasmid-mediated quinolone and aminoglycoside resistance genes in Escherichia coli inhabiting a major anthropogenic river of India.

Abstract Presence of antimicrobial resistance (AMR) genes in Escherichia coli inhabiting anthropogenic rivers is an important public health concern because plasmid-mediated AMR genes can easily spread to other pathogens by horizontal gene transfer. Besides β-lactams, quinolones and aminoglycosides are the major antibiotics against E. coli. In the present study, we have investigated the presence of plasmid-mediated quinolone resistance (PMQR) and aminoglycoside resistance genes in E. coli isolated from a major river of northern India. Our results revealed that majority of the strains were phenotypically susceptible for fluoroquinolones and some aminoglycosides like amikacin, netilmicin, tobramycin and gentamicin. However, 16.39% of the strains were resistant for streptomycin, 8.19% for kanamycin and 3.30% for gentamicin. Of the various PMQR genes investigated, only qnrS1 was present in 24.59% of the strains along with ISEcl2. Aminoglycoside-resistance genes like strA-strB were found to be present in 16.39%, aphA1 in 8.19% and aacC2 in only 3.30% of the strains. Though, no co-relation was observed between phenotypic resistance for fluorquinolones and presence of PMQR genes, phenotypic resistance for streptomycin, kanamycin and gentamicin exactly co-related with the presence of the genes strA-strB, aphA1 and aacC2, respectively. Moreover, all the AMR genes discerned in aquatic E. coli were found to be situated on conjugative plasmids and, thus easily transferrable. Our study accentuates the importance of routine surveillance of urban rivers to curtail the spread of AMR genes in aquatic pathogens.

Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal.

The present study aimed to characterize the extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases (ESBL/PMAβ) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAβ [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6’)-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

Seabirds as anthropization indicators in two different tropical biotopes: A One Health approach to the issue of antimicrobial resistance genes pollution in oceanic islands

Antimicrobial resistance is a quintessential One Health issue, among the most serious 21st century global threats to human health. Seabirds may act as sentinels of natural and anthropogenic changes in the marine ecosystem health, including pollution by antimicrobial resistance genes (ARGs). We used real time PCR to identify and quantify 22 plasmid-mediated ARGs in the gastrointestinal microbiome of six wild seabird species, comparing an anthropized (Fernando de Noronha Archipelago - FNA) and a pristine biotope (Rocas Atoll - ROA), Brazil. Of 257 birds, 218 (84.8%) were positive to at least one ARG. ARG classes encoding resistance to tetracyclines (75.1%), quinolones (10.5%) and phenicols (10.5%) were the most prevalent, with tetracyclines significantly greater than the remaining classes (p < 0.05). Genes tet(S) (29.2%), tet(A) (28.8%), and tet(B) (24.9%) were the most commonly found and had a significantly greater prevalence when compared to the remaining ARGs (p < 0.05). The anthropized biotope presented statistically significant higher prevalence of sulfonamide- and quinolone-encoding ARGs in comparison with the pristine (respectively, p = 0.01 and p = 0.03), and higher sulII gene prevalence (p = 0.04), consistent with anthropogenic pressure. Migratory species (only present in ROA) showed statistically significant higher mcr-1 (polymyxins) and blaTEM (betalactam) prevalences (respectively, p = 0.009 and p = 0.02), and mcr-1 percentage load (p = 0.0079) in comparison with non-migratory. To our knowledge, this is the largest ARGs survey based on direct detection and quantification in seabirds worldwide, and the first to evaluate non-synanthropic species in oceanic islands. This is the first detection of mcr-1 in wild free-ranging seabirds in Brazil and in free-ranging migratory non-synanthropic seabirds worldwide. Our findings show the importance of biological and ecological factors, highlighting the role of seabirds as anthropization sentinels and ARGs-pollution environmental indicators (even in a pristine biotope), and their involvement in the One Health epidemiological chain of ARGs.

Molecular characterization of antimicrobial resistance genes on farms and in commercial milk with emphasis on the effect of currently practiced heat treatments on viable but nonculturable formation

Despite the considerable advances that have been made to improve dairy food safety, there is rising concern that pasteurization is not sufficient for the destruction of plasmid-mediated antimicrobial resistance (AMR) genes of resistant bacteria and could stimulate bacteria to enter into a viable but nonculturable (VBNC) state. In the current study, we surveyed the prevalence of 1 genomic and 9 plasmid-mediated AMR genes in 100 samples (bulk tank milk and milk filter socks) at the farm level and 152 commercial milk samples (pasteurized and UHT milks) and assessed the VBNC state in dairy bacteria. Results revealed that sul2 was the most prevalent plasmid-mediated gene in milk filter socks (96%), bulk tank milk (48%), pasteurized milk (68%), and UHT (43%) milk; in contrast, mecA was not detected in any sample. Additionally, commercial pasteurization (as currently practiced) failed to decrease the prevalence of the blaTEM-B1 (43%), tetK (30%), and tetA (55%) plasmid-mediated AMR genes; thus, commercial pasteurization may be one of the factors creating the VBNC state in some dairy bacteria. Continued research is necessary to identify bacterial species entering the VBNC state after pasteurization, to assess their potential hazard level and shed more light on the expression and possibility of horizontal gene transfer of those plasmid-mediated AMR genes.

Preservatives accelerate the horizontal transfer of plasmid-mediated antimicrobial resistance genes via differential mechanisms

Increasing concentrations of preservatives have been detected in environments due to the overuse and misuse of preservatives in food and personal care products. Recent studies have relied heavily on the toxicity, biodegradability, and fate of preservatives in the environment. However, the biological effects of preservatives on antimicrobial resistance, which poses great threats to public health worldwide, are largely unknown. This study investigated three preservatives for their ability and mechanisms of promoting horizontal transfer of antimicrobial resistance genes (ARGs). The results demonstrated that these preservatives (sodium nitrite, sodium benzoate, and triclocarbon), under daily-use concentrations, led to concentration-dependent increases in conjugative transfer by 1.24–2.63, 6.79–7.05, and 2.17–4.31 folds compared with the control group. Even these three preservatives had different patterns on generating intracellular reactive oxidative species (ROS) and reactive nitrogen species (RNS), all of them could stimulate radical-induced RpoS regulon and SOS response, increase cell membrane permeability, and regulate conjugative transfer-related genes, subsequently promoting horizontal transfer of ARGs. The present results expanded the understanding of biological effects induced by preservatives, and provided mechanistic insight into the preservatives-induced resistance. This study also opens an intriguing question on the roles of emerging contaminants including preservatives in the emerging and spread of ARGs in various environments.

Antimicrobial resistance genes present in the faecal microbiota of free-living Galapagos tortoises (Chelonoidis porteri).

Antimicrobial resistance (AMR), encoded by plasmid-mediated AMR genes (ARGs), is an increasing global public health threat. Wildlife play a fundamental role as sentinels, reservoirs and potential vectors of ARGs. For the first time in Galapagos, we have identified and quantified the presence of ARGs in free-living giant tortoises (Chelonoidis porteri). We performed ARG analyses by quantitative PCR of faeces collected from the cloaca of 30 tortoises widely distributed across Santa Cruz Island. Validated samples (n=28) were analysed by a panel of up to 21 different ARGs and all 28 tortoise samples were positive to one or more genes encoding resistance. Thirteen of 21 tested ARGs were present in at least one sample, and 10 tortoises (35.7%) had a multi-resistant pattern. We recommend additional research so we may more fully understand resistance patterns across taxa and geographical locations throughout the Galapagos archipelago, and the implications of ARGs for the health of wildlife, domestic animals, and humans. In this study, we found 100% of sampled giant tortoises had ARGs present in their faeces, suggesting a large-scale distribution of these genes within the archipelago.

The effect and mechanism of urban fine particulate matter (PM2.5) on horizontal transfer of plasmid-mediated antimicrobial resistance genes

Fine particulate matter (PM2.5) and antimicrobial resistance are two major threats to public health worldwide. Current air pollution studies rely heavily on the assessment of PM2.5 chemistry and toxicity. However, whether and how PM2.5 affects the proliferation and transfer of antimicrobial resistance genes (ARGs) in various environments has remained unanswered. This study investigated the effects and potential mechanisms of urban PM2.5 on the horizontal transfer of ARGs between opportunistic Escherichia coli (E. coli) strains. The results showed that urban PM2.5 samples collected from Xi'an (XA), Shanghai (SH), and Shijiazhuang (SJZ) in China induced location- and concentration-dependent promotion of conjugative transfer frequencies compared to the control group. The relevant mechanisms were also explored, including the formation of intracellular reactive oxygen species (ROS) and the subsequent induction of oxidative stress, SOS response, changes in membrane permeability, and alternations in mRNA expression of genes involved in horizontal transfer. This study highlights the effect of PM2.5 on promoting the horizontal transfer of ARGs and elucidates the mechanism of the antimicrobial-resistance risks posed by urban PM2.5. These findings are of great value in understanding the transmission of antimicrobial resistance in various environments and provide valuable information for re-evaluating air quality assessment practices.

Petrol and diesel exhaust particles accelerate the horizontal transfer of plasmid-mediated antimicrobial resistance genes

Particles exhausted from petrol and diesel consumptions are major components of urban air pollution that can be exposed to human via direct inhalation or other routes due to atmospheric deposition into water and soil. Antimicrobial resistance is one of the most serious threats to modern health care. However, how the petrol and diesel exhaust particles affect the development and spread of antimicrobial resistance genes (ARGs) in various environments remain largely unknown. This study investigated the effects and potential mechanisms of four representative petrol and diesel exhaust particles, namely 97 octane petrol, 93 octane petrol, light diesel oil, and marine heavy diesel oil, on the horizontal transfer of ARGs between two opportunistic Escherichia coli (E. coli) strains, E. coli S17-1 (donor) and E. coli K12 (recipient). The results demonstrated that these four representative types of nano-scale particles induced concentration-dependent increases in conjugative transfer rates compared with the controls. The underlying mechanisms involved in the accelerated transfer of ARGs were also identified, including the generation of intracellular reactive oxygen species (ROS) and the consequent induction of oxidative stress, SOS response, changes in cell morphology, and the altered mRNA expression of membrane protein genes and those involved in the promotion of conjugative transfer. The findings provide new evidences and mechanistic insights into the antimicrobial resistance risks posed by petrol and diesel exhaust particles, and highlight the implications and need for stringent strategies on alternative fuels to mitigate air pollution and health risks.

Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat.

The dissemination of plasmid-mediated antimicrobial resistance genes may pose a substantial public health risk. In the present work, the occurrences of blaCTX-M and plasmid-mediated ampC and qnr genes were investigated in Escherichia coli from 16 chicken carcasses produced by four commercial brands in Brazil. Of the brands tested, three were exporters, including one of organic chicken. Our study assessed 136 E. coli isolates that were grouped into 77 distinct biotypes defined by their origin, resistance profiling, the presence of β-lactamase and plasmid-mediated quinolone resistance genes and enterobacterial repetitive intergenic consensus-polimerase chain reaction typing. The blaCTX-M-15, blaCTX-M-2 and blaCTX-M-8 genes were detected in one, 17 and eight different biotypes, respectively (45 isolates). Twenty-one biotypes (46 isolates) harboured blaCMY-2. Additionally, blaCMY-2 was identified in isolates that also carried either blaCTX-M-2 or blaCTX-M-8. The qnrB and/or qnrS genes occurred in isolates carrying each of the four types of β-lactamase determinants detected and also in oxyimino-cephalosporin-susceptible strains. Plasmid-mediated extended-spectrum β-lactamase (ESBL) and AmpC determinants were identified in carcasses from the four brands tested. Notably, this is the first description of blaCTX-M-15 genes in meat or food-producing animals from South America. The blaCTX-M-8, blaCTX-M-15 and blaCMY-2 genes were transferable in conjugation experiments. The findings of the present study indicate that plasmid-mediated ESBL and AmpC-encoding genes are widely distributed in Brazilian chicken meat.

Comparison of antimicrobial resistance phenotypes and genotypes in enterotoxigenic Escherichia coli isolated from Australian and Vietnamese pigs.

This study aimed to compare the antibiogram phenotype and carriage of antimicrobial resistance genes (ARGs) of 97 porcine multidrug-resistant (MDR) enterotoxigenic Escherichia coli (ETEC) isolates obtained from Vietnam and 117 porcine MDR-ETEC obtained from Australia, two countries with different antimicrobial regulation systems. An antimicrobial resistance index (ARI) was calculated to quantify their potential significance to public health. Both Vietnamese and Australian isolates had moderate to high levels of resistance to commonly used antibiotics (ampicillin, tetracycline and sulphonamides). None of the Australian isolates were resistant to fluoroquinolones or third-generation cephalosporins and none possessed associated plasmid-mediated ARGs. However, 23.1% of Australian isolates were resistant to gentamicin owing to ARGs associated with apramycin or neomycin resistance [e.g. aac(3)-IV] that impart cross-resistance to gentamicin. Whilst Vietnamese isolates carried aminoglycoside ARGs, 44.4% of commercial pig isolates were resistant to gentamicin in comparison with 0% of village pig isolates. The plasmid-mediated fluoroquinolone ARG qnrB was commonly detected in Vietnamese isolates (52.3% commercial, 44.1% village), but phenotypic resistance was low (3.2% and 11.8%, respectively). The mean ARI for Vietnamese isolates (26.0) was significantly different (P<0.001) from the mean ARI for Australian isolates (19.8), primarily reflecting fluoroquinolone resistance in the former collection. This comparison suggests the effectiveness of regulations that slow the dissemination of 'critical' resistance by restricting the availability of important classes of antimicrobials.

Characterization of CTX-M-14- and CTX-M-15-Producing Escherichia coli and Klebsiella pneumoniae Isolates from Urine Specimens in a Tertiary-Care Hospital

This study aimed to characterize CTX-M producers of urinary E. coli and K. pneumoniae isolates and to determine the prevalence of plasmid-mediated antimicrobial resistance genes among them. Minimum inhibitory concentrations (MICs) were determined, and PCR and sequencing were performed. Among the 42 (82.3%) E. coli and 24 (77.4%) K. pneumoniae isolates containing bla(CTX-M), bla(CTX-M-14) and bla(CTX-M-15) were detected in 23 and 19 E. coli isolates, respectively, and in 7 and 17 K. pneumoniae isolates, respectively. CTX-M producers of urinary E. coli and K. pneumoniae were resistant to multiple antibiotics and contained other antimicrobial resistance genes. CTX-M-15 producers contained more antimicrobial resistance genes than did CTX-M-14 producers.

Prevalence of antiseptic resistance genes qacA/B and specific sequence types of methicillin-resistant Staphylococcus aureus in the era of hand hygiene

Sir, In an attempt to control the spread of methicillin-resistant Staphylococcus aureus (MRSA) and other multiresistant organisms in hospitalized patients, hand hygiene campaigns have promoted the widespread use of alcohol-based hand solutions containing antiseptic agents such as chlorhexidine and quaternary ammonium compounds. The use of these products, however, has led to increased resistance to these antiseptic agents in MRSA strains. – 5 qacA/B are plasmid-borne resistance genes coding for multidrug efflux pumps that confer high-level resistance to chlorhexidine, quaternary ammonium compounds and other antiseptic compounds commonly used in hand hygiene solutions. They are associated with higher MICs and tolerance of these antiseptic agents. qacA/B genes have been found in healthcare-associated MRSA (HA-MRSA) isolates from around the world. – 8 In addition, the prevalence of qacA/B genes has been associated with particular sequence types (STs) of HA-MRSA. To our knowledge, the prevalence of MRSA strains harbouring these genes has not been researched in Australia. In this study, we characterize the prevalence of qacA/B genes in HA-MRSA isolates in our institution spanning 10 years, from 2000 to 2009. We determined whether the national hand hygiene campaign implemented in 2006 had any impact on the prevalence of these genes and, in addition, if any relationship exists between the different STs of HA-MRSA and qacA/B gene prevalence. All (total 151) clinically significant unique HA-MRSA isolates from the period 2000–09 from Nepean Hospital, a 490 bed tertiary referral hospital in Sydney, were used in this study. The presence of qacA/B genes in these isolates was evaluated by real-time PCR using the following primers: qacA/B forward primer 5′-CTATGGCAATAGGAGATATGGTGT and reverse primer 5′-CCACTACAGATTCTTCAGCTACATG. The HA-MRSA isolates were categorized into STs using their antibiograms and correlations with pre-existing typing performed on HA-MRSA isolates in our institution by the Australian Group on Antimicrobial Resistance. Quantities of hand hygiene solutions used by the hospital were obtained via the pharmacy database. A sustained .6-fold increase in antiseptic-containing alcoholbased hand solution use occurred as a result of the hand hygiene campaign (‘Clean hands saves lives’) implemented in 2006 [amounts of 0.5% chlorhexidine gluconate and quaternary ammonium compounds used (average litres per year) were 968 and 150 L before the hand hygiene campaign (2004–06) and 3967 and 2958 L after the hand hygiene campaign (2007–09), respectively]. The yearly prevalence of qacA/B genes in HA-MRSA isolates from 2000 to 2009 ranged from 65.0% to 94.7% (mean 78.6%). This is higher than that previously found in the UK (8%–26%), Europe (63%) and Asia (33%–61%), but comparable to that found in a recent study in Geneva (79%). The HA-MRSA isolates in this study comprise two STs: ST239 MRSA-III Aus-2 EMRSA and ST22 MRSA-IV EMRSA-15. The ST239 type was the predominant strain throughout the study period (mean yearly prevalence of ST239 from 2000 to 2009 was 84.3%, range 75%–100%). Comparing the prevalence of the qacA/B genes before (2000– 06) and after (2007–09) the hand hygiene campaign, we did not find an increase in the prevalence of these genes despite the marked increase in antiseptic hand solutions after 2006 [mean yearly prevalence of qacA/B-positive MRSA isolates between 2000 and 2006 and between 2007 and 2009 was 78.6% and 73.5%, respectively (P1⁄40.53; x1⁄40.39, df1⁄41)]. Similarly, there was no change in the incidence of MRSA ST239 [mean yearly prevalence of MRSA ST239 between 2000 and 2006 and between 2007 and 2009 was 85.6% and 78.6%, respectively (P1⁄40.37; x1⁄40.81, df1⁄41)]. Interestingly, we found a significant association between qacA/B gene positivity and MRSA type ST239 [94.5% of qacA/Bpositive isolates were ST239 and 88.9% of ST239 isolates were qacA/B positive (OR1⁄429.09, CI1⁄48.93–94.82)]. This relationship between qacA/B and MRSA ST239 has been suggested in previous studies in the UK and Taiwan; however, the prevalence of this MRSA strain, as well as qacA/B genes, was much lower in these studies. MRSA ST239 is found in significantly high numbers in Eastern Australia, and is associated with a broad spectrum of antimicrobial resistance and epidemic potential. The qacA/B genes are thought to be located on multiresistance plasmids, such as pSK1, and co-transmitted with plasmid-mediated antimicrobial resistance genes; hence their association with this multiresistant strain of MRSA is logical. The high prevalence of this ST239 strain could be due to selective pressure from the use of antiseptic products and/or the widespread use of antimicrobial agents. Our findings suggest the latter, given the absence of a corresponding rise in qacA/B genes with increased antiseptic hand solution use; however, the baseline use of antiseptic agents within the hospital could also explain the high prevalence of qacA/B genes even prior to the hand hygiene campaign.

Antimicrobial resistance and virulence gene profiles in multi-drug resistant enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea

This study aimed to characterize antimicrobial resistance and virulence genes in multi-drug resistant enterotoxigenic Escherichia coli (ETEC) isolates ( n = 117) collected from porcine post-weaning diarrhoea cases in Australia (1999–2005). Isolates were serotyped, antibiogram-phenotyped for 12 antimicrobial agents and genotyped by PCR for 30 plasmid-mediated antimicrobial resistance genes (ARGs), 22 intestinal and 38 extraintestinal E. coli virulence genes (VGs). Nine serogroups were identified, the most prevalent being O149 (46.2%), O141 (11.2%) and Ont (31.6%). None of the isolates showed resistance to ceftiofur or enrofloxacin and 9.4% were resistant to florfenicol. No corresponding extended-spectrum/AmpC β-lactamase, fluoroquinolone or floR ARGs were detected. An antimicrobial resistance index (ARI) was calculated from the combined data with a weighting for each antimicrobial agent dependent upon its significance to human health. Serogroup O141 isolates had a significantly higher ARI due to an elevated prevalence of aminoglycoside ARGs and possession of more virulence genes (VGs), including ExPEC or EHEC adhesins ( bmaE, sfa/focDE, fimH, ihA) in toxin-producing strains that lacked the normally associated F4 and F18 fimbriae. Few associations between ARGs and VGs were apparent, apart from tetC, sfa/focDE and ompT which, for a sub-set of O141 isolates, suggest possible plasmid acquisition from ExPEC. The multi-drug resistant ETEC ARG/VG profiles indicate a high probability of considerable strain and plasmid diversity, reflecting various selection pressures at the individual farm level rather than emergence and lateral spread of MDR resistant/virulent clones.

Identification of blaCMY-7 and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia

To determine clonality and identify plasmid-mediated resistance genes in 11 multidrug-resistant Escherichia coli (MDREC) isolates associated with opportunistic infections in hospitalized dogs in Australia. Phenotypic (MIC determinations, modified double-disc diffusion and isoelectric focusing) and genotypic methods (PFGE, plasmid analysis, PCR, sequencing, Southern hybridization, bacterial conjugation and transformation) were used to characterize, investigate the genetic relatedness of, and identify selected plasmid-mediated antimicrobial resistance genes, in the canine MDREC. Canine MDRECs were divided into two clonal groups (CG 1 and 2) with distinct restriction endonuclease digestion and plasmid profiles. All isolates possessed bla(CMY-7) on an approximately 93 kb plasmid. In CG 1 isolates, bla(TEM), catA1 and class 1 integron-associated dfrA17-aadA5 genes were located on an approximately 170 kb plasmid. In CG 2 isolates, a second approximately 93 kb plasmid contained bla(TEM) and unidentified class 1 integron genes, although a single CG 2 strain carried dfrA5. Antimicrobial susceptibility profiling of E. coli K12 transformed with CG 2 large plasmids confirmed that the bla(CMY-7)-carrying plasmid did not carry any other antimicrobial resistance genes, whereas the bla(TEM)/class 1 integron-carrying plasmid carried genes conferring resistance to tetracycline and streptomycin also. This is the first report on the detection of plasmid-mediated bla(CMY-7) in animal isolates in Australia. MDREC isolated from extraintestinal infections in dogs may be an important reservoir of plasmid-mediated resistance genes.