Pulsed-field Gel Electrophoresis Fingerprints Research Articles

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

Antimicrobial resistance and biofilm formation capacity among Acinetobacter baumannii strains isolated from patients with burns and ventilator-associated pneumonia.

Acinetobacter baumannii is a pathogen responsible for nosocomial infections, especially in patients with burns and ventilator-associated pneumonia (VAP). The aims of this study was to compare the biofilm formation capacity, antimicrobial resistance patterns and molecular typing based on PFGE (Pulsed-Field Gel Electrophoresis) in A.baumannii isolated from burn and VAP patients. A total of 50 A.baumannii isolates were obtained from burn and VAP patients. In this study, we assessed antimicrobial susceptibility, biofilm formation capacity, PFGE fingerprinting, and the distribution of biofilm-related genes (csuD, csuE, ptk, ataA, and ompA). Overall, 74% of the strains were multidrug resistant (MDR), and 26% were extensively drug-resistant (XDR). Regarding biofilm formation capacity, 52%, 36%, and 12% of the isolates were strong, moderate, and weak biofilm producers. Strong biofilm formation capacity significantly correlated with XDR phenotype (12/13, 92.3%). All the isolates harbored at least one biofilm-related gene. The most prevalent gene was csuD (98%), followed by ptk (90%), ataA (88%), ompA (86%), and csuE (86%). Harboring all the biofilm-related genes was significantly associated with XDR phenotype. Finally, PFGE clustering revealed 6 clusters, among which cluster No. 2 showed a significant correlation with strong biofilm formation and XDR phenotype. Our findings revealed the variable distribution of biofilm-related genes among MDR and XDR A.baumannii isolates from burn and VAP patients. A significant correlation was found between strong biofilm formation capacity and XDR phenotype. Finally, our results suggested that XDR phenotype was predominant among strong-biofilm producer A.baumannii in our region.

Drug Resistance and Homology of Pathogens of Infective Diarrhea in East Heilongjiang, China.

ObjectiveTo investigate drug resistance and homology of pathogens that can cause infective diarrhea in East Heilongjiang, China, and provide suitable references for the clinical treatment.MethodsThe process of isolation and identification of pathogenic bacteria in the faeces and blood of diarrhea patients was conducted. The drug resistance of the isolated pathogenic bacteria was analyzed by VITEK 2 compact drug sensitivity analysis system, and the strain homology was determined by pulsed field gel electrophoresis (PFGE).ResultsFrom 2014 to 2018, 28 different strains of pathogenic bacteria were isolated from 657 samples of faeces and blood, including 23 strains of Salmonella, 3 strains of Vibrio parahaemolyticus and 2 strains of diarrheal Escherichia coli, with detection rates of 3.5%, 0.46% and 0.30% respectively. Among them, 22 strains of Salmonella exhibited significant drug resistance, with a drug resistance rate of 95.65%. A total of 11 different PFGE fingerprints of 23 Salmonella strains were also obtained, with a homology of 56.5–100%.ConclusionAmong the various pathogens causing infective diarrhea in East Heilongjiang, China, Salmonella displayed multiple drug resistance and its distribution was found to be polymorphic.

Pulsed-field gel electrophoresis fingerprinting of Listeria monocytogenes isolates recovered from foods of animal origin and fishes in North-Eastern India.

L isteria monocytogenes is a pathogen of great concern to the food industry. The present study was aimed to explore the clonal relationships amongst L. monocytogenes strains isolated from foods of animal origin (milk, beef, chevon (goat meat), pork and chicken) and fish. Forty-seven L. monocytogenes strains were characterized by pulsed-field gel electrophoresis (PFGE). The PFGE analysis using ApaI and AscI enzymes revealed 37 pulsotypes, with Simpson’s discriminatory index of 0.987. This study demonstrated the presence of a few similar L. monocytogenes pulsotypes in different foods of animal origin in different places and years of isolation and this indicates that some L. monocytogenes subtypes may be ubiquitous which are acclimatizing and persisting in different foods of animal origin. This also emphasizes the importance of cross-contamination in local wet markets. Thus, the understanding of genetic diversity will contribute to the development of rational and workable strategies to control this important zoonotic infection.

Determination of the ciprofloxacin-resistant Escherichia coli isolated from chicken meat in Turkey

In this study, the occurrence of the ciprofloxacin-resistant (CR) Escherichia coli in chicken meat was determined, and their clonal relations were investigated by using pulsed-field gel electrophoresis (PFGE). Antimicrobial resistance patterns of E. coli isolates were determined by using disc diffusion assay, and minimum inhibitory concentration of ciprofloxacin was determined by E-test. Plasmid-mediated quinolone resistance (PMQR) and extended spectrum beta-lactamase (ESBL) resistance genes were also screened through polymerase chain reactions. Sixty chicken meat samples were collected from different supermarkets and butchers in Sivas, Turkey. CR E. coli strains were determined in 59 (98.3%) chicken meat samples. By analyzing PFGE fingerprint data, 34 different pulsotypes were determined. All E. coli strains were found to be resistant to nalidixic acid, enrofloxacin, and norfloxacin. In addition, isolates were resistant to levofloxacin (40.7%), ampicillin (94.9%), trimethoprim-sulfamethoxazole (76.3%), tetracycline (69.5%), and chloramphenicol (44.1%). However, isolates were susceptible to imipenem and colistin. In this study, 81.4% of CR E. coli isolates were observed to have a multidrug-resistant profile, which is defined as resistance to three or more classes of antibiotics. Through phenotypic confirmation tests, five isolates (8.3%) were determined to be ESBL-producing. The PMQR genes were not determined in any of the isolates. Two isolates (3.4%) possessed the blaCTX-M and blaCMY-2 genes, and 40 isolates (67.8%) had the blaTEM gene. Taken together, retail raw chicken meat is highly contaminated with CR E. coli. However, these isolates are not found to be carriers of the PMQR genes, indicating a low public health problem.

Homology analysis of 51 penicillin-intermediate Streptococcus pneumoniae isolates from Wenzhou City, China.

ObjectiveTo investigate drug resistance features and homology among penicillin-intermediate Streptococcus pneumoniae isolates from Wenzhou City, China.MethodsFifty-one penicillin-intermediate S. pneumoniae isolates were obtained from respiratory samples of infants and children hospitalized with lung infections. An antimicrobial susceptibility test was used to assess drug resistance. Polymerase chain reaction and agarose gel electrophoresis were used to identify S. pneumoniae isolates and pulsed-field gel electrophoresis (PFGE) was used to analyze molecular subtypes. Hierarchical cluster analysis of PFGE fingerprints was used to compare genetic diversity and relatedness of S. pneumoniae isolates. The Quellung test was used for serotyping.ResultsFifty-one penicillin-intermediate S. pneumoniae isolates showed evidence of multi-drug resistance and polyclonal origins. The isolates were classified into 25 subtypes through hierarchical cluster analysis of PFGE fingerprints. Three of these subtypes formed a supertype (15/51, 16/51 and 8/51 isolates), while the remaining subtypes occurred sporadically (12/51 isolates).ConclusionsTransmission of penicillin-intermediate S. pneumoniae is mostly vertical and to a lesser extent horizontal. Effective prevention strategies, including respiratory tract management and contact isolation, are essential to control nosocomial S. pneumoniae infection. Once susceptibility is confirmed, vancomycin, high-dose penicillin or third-generation cephalosporins (cefotaxime and ceftriaxone) may be used to treat penicillin-intermediate S. pneumoniae.

Molecular epidemiological analysis of carbapenem-resistant Klebsiella pneumonia in China from 2009 to 2016

Objective The study aimed to investigate the drug sensitivity and molecular epidemiological analysis of carbapenem-resistant Klebsiella pneumoniae in China from 2009 to 2016. Methods For Klebsiella pneumoniae collected from China Antimicrobial Resistance Surveillance Trial (CARST) from 2009 to 2016, those resist to ertapenem were screened out as the carbapenem-resistant Klebsiella pneumoniae(CRKP). Antibiotic susceptibilities of 14 antibacterial agents to the bacteria were assessed by minimum inhibitory concentration (MIC) using two-fold agar dilution test which was recommended by the Clinical and Laboratory Standard Institute (CLSI). Carbapenemases were filtered out by the modified carbapenem inactivation method(mCIM) and EDTA-modified carbapenem inactivation method(eCIM) test, PCR was used to screen for the presence of carbapenemase genes or not. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homology of these isolates. Results A total of 193 CRKP were screened out in the 2 502 Klebsiella pneumoniae isolates. The resistance rates of 193 CRKP isolates to colistinE (4.0%), tigecycline (7.2%), fosfomycin (25.4%) and amikacin (38.3%) were less than 40%. However, the resistance rates to the other 9 antimicrobial agents were higher than 65%. There were 143 carbapenemase gene detected in 193 CRKP, of which blaKPC-288(45.6%) and blaNDM-140(20.7%) were the most common. In the 126 isolates of Klebsiella pneumoniae producing KPC-2 and NDM-1 carbapenemases, a total of 33 ST-types were detected, mainly was ST11 (59.5%). All ST11 strains produce KPC-2 carbapenemase. A total of 111 clones were obtained from PFGE fingerprints, there were 12 clones in which the same clone strains were equal or greater than 2. Conclusion The resistance rate of Klebsiella pneumoniae to carbapenems has been rising from 2009 to 2016, and clinicians should take reasonable measures to strengthen the monitoring of resistance to CRKP and control its spread across the country. Key words: Klebsiella pneumoniae; Carbapenems; Drug resistance, bacterial; Microbial sensitivity tests; Electrophoresis, gel, pulsed-field; Multilocus sequence typing

Pulsed-field gel electrophoresis fingerprinting of Campylobacter jejuni and Campylobacter coli strains isolated from clinical specimens, Iran.

The aim of this study was to determine the clonal correlation of Campylobacter strains isolated from diarrheal children under 5years of age in Iran using the PFGE method and to determine the antimicrobial susceptibility and virulence gene content of strains. Of 750 patients with bacterial diarrhea, 33 (4%) Campylobacter spp., including 31 C. jejuni (94%) and 2 C. coli (6%), were isolated during 18-month period in Tehran, Iran. Except for one strain, remaining Campylobacter strains were positive for the flaA gene. A complete set of cytolethal distending toxin (CDT) encoding genes (cdtABC) were detected in 52% of the C. jejuni strains, while the 2 C. coli isolates under study only harbored cdtA and cdtB of the CDT cluster. All strains were resistant to at least three antibiotic classes. Resistance to ampicillin among C. coli and C. jejuni strains was 100% and 84%, respectively, and 80% of all strains were susceptible to gentamicin. PFGE genotyping generated 19 pulsotypes with two major clusters, displaying the maximum and minimum similarity of 100% and 26%, respectively. The C. coli strains showed clearly distinct pulsotypes and each fell within separate clusters. A very homogeneous Campylobacter population was detected among Iranian patients with 33 % of strains showing identical banding patterns and no clear correlation was observed between antibiotic resistance profiles and PFGE patterns of the isolates.

The population structure of Ochrobactrum isolated from entomopathogenic nematodes indicates interactions with the symbiotic system

Entomopathogenic nematodes (EPNs) form specific mutualistic associations with bioluminescent enterobacteria. In Heterorhabditidis indica, Ochrobactrum spp. was identified beside the symbiont Photorhabdus luminescens but its involvement in the symbiotic association in the EPNs remains unclear. This study describe the population structure and the diversity in Ochrobactrum natural populations isolated from EPNs in the Caribbean basin in order to question the existence of EPN-specialized clones and to gain a better insight into Ochrobactrum-EPNs relationships. EPN-associated Ochrobactrum and Photorhabdus strains were characterized by multi-locus sequence typing, Pulsed-Field Gel Electrophoresis fingerprinting and phenotypic traits.Population study showed the absence of EPN-specialized clones in O. intermedium and O. anthropi but suggested the success of some particular lineages. A low level of genetic and genomic diversification of Ochrobactrum isolated from the natural population of Caribbean nematodes was observed comparatively to the diversity of human-associated Ochrobactrum strains. Correspondences between Ochrobactrum and P. luminescens PFGE clusters have been observed, particularly in the case of nematodes from Dominican Republic and Puerto Rico. O. intermedium and O. anthropi associated to EPNs formed less biofilm than human-associated strains.These results evoke interactions between Ochrobactrum and the EPN symbiotic system rather than transient contamination. The main hypothesis to investigate is a toxic/antitoxic relationship because of the ability of Ochrobactrum to resist to antimicrobial and toxic compounds produced by Photorhabdus.

Mother-to-child transmission of extended-spectrum-beta-lactamase-producing Enterobacteriaceae

Preterm infants are at high risk for extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) sepsis and neonatal intensive care unit (NICU) outbreaks. Maternal colonization with ESBL-E may be precursory to mother-to-child transmission. However, there is no consensus regarding surveillance of pregnant women for ESBL-E colonization. To identify pairs of mothers and infants harbouring same-strain ESBL-E colonization and to determine whether maternal transmission may play a role in increasing ESBL-E carriage in preterm infants. This was a one-year analysis from an ongoing, prospective ESBL-E surveillance of mothers of premature infants and their offspring. Mother-infant pairs colonized with the same bacteria underwent strain analysis using pulsed-field gel electrophoresis (PFGE). Clinical parameters were collected from the hospital computerized records. Between January 2015 and January 2016, 313/409 (76.5%) mothers and all 478 (100%) infants were screened for ESBL-E colonization; carriage rates were 21.5% and 14.8%, respectively. Four (5.6%) colonized infants developed late-onset sepsis and two (2.8%) died. Twenty-five mother-infant pairs colonized with the same bacterial strain were identified; a subgroup of 10 pairs of isolates underwent PFGE, and 70% displayed an identical PFGE fingerprint. No similarities were found between isolates recovered from unrelated neonates and mothers. ESBL-E colonization was found significantly earlier in infants of mothers colonized at birth (P<0.001) compared with infants of non-colonized mothers. ESBL-E carriage rates in mothers and NICU infants with non-negligible maternal-neonatal ESBL-E transmission in the study region indicate that maternal colonization surveillance and/or further infection control interventions should be considered.

A Retrospective Study of Salmonella Enteritidis Isolated from Commercial Layer Flocks.

Consumption of shell eggs has been associated with Salmonella Enteritidis (SE) infections in humans in the United States. Because of this, the Pennsylvania Egg Quality Assurance Program (PEQAP) was developed and implemented in 1994. The PEQAP involves periodic flock testing and management practices to minimize SE contamination of shell eggs. Subsequently, the U.S. Food and Drug Administration (FDA) introduced a mandatory federal program in 2010 and 2012 for shell egg producers modeled closely after PEQAP to reduce the incidence and prevalence of SE during production, storage, and transport nationwide. In this study, a retrospective epidemiologic analysis was conducted by characterizing SE isolated from commercial layer environment samples and shell eggs submitted to the Animal Diagnostic Laboratory at The Pennsylvania State University using phage typing and pulsed-field gel electrophoresis (PFGE). The objective of this study was to determine the relatedness of SE isolates from hen house environments and shell eggs and to optimize the existing protocols of egg quality assurance programs by identifying the best layer-house environmental sampling time points in order to minimize SE contamination of shell eggs. A total of 94 SE isolates from 65 hen flocks on 35 premises in Pennsylvania recovered during 2007 to 2015 were used in this study. The SE phage type 8 and PFGE fingerprint type JEGX01.0004 most commonly associated with human SE infection was also the predominant type present in layer-house environments and shell eggs. This reconfirms hen house environmental monitoring is an effective method to identify SE-infected flocks. Further, the PEQAP program allowed SE detection of infected flocks earlier than the FDA program as it included an additional environmental test at 29-31 wk of age, enabling the earlier prevention of SE-contaminated shell eggs going to the market. Therefore, it is recommended to refine the sampling time points of the current FDA Egg Rule by adding hen house environmental testing at 29-31 wk of age.

Dissemination of the Methicillin Resistant Staphylococcus aureus (MRSA) Pediatric Clone (ST5-T002-IV-Pvl+) as a Major Cause of Community Associated (CA) Staphylococcal Infections in Bedouin Children, Southern Israel

BackgroundPediatric CA-MRSA infections are emerging worldwide. High CA-MRSA carriage rates were previously described in healthy Bedouin children (Adler et al, J Clin Microbiol 2009). We assessed demographic, clinical and molecular characteristics of MRSA infections in children in southern Israel.MethodsSoroka University Medical Center microbiology laboratory serves the entire population of southern Israel, divided into two ethnic groups, Bedouin and Jews. All in-hospital MRSA isolates from children 0–18 years, obtained in 2016 were included.Clinical data were recorded from the hospital’s computerized records. Health-care associated (HA) and community-associated infections were defined according to the US Center for Disease Control and Prevention. All isolates were evaluated for staphylococcal cassette chromosome (SCCmec), Panton–Valentine leucocidin (PVL), Staphylococcus aureus protein A (spa) type as well as by pulsed-field-gel-electrophoresis (PFGE) and antimicrobial susceptibility testing.ResultsOverall 95 (18%) of S. aureus isolates were MRSA (Table 1). Twenty-five different MRSA strains were identified. 28 isolates (29.5% of all MRSA) belonged to a pediatric clone, rarely observed in Israel (SCC IV, PVL positive, spa type 002; all demonstrate identical PFGE fingerprints). 82% of infections caused by this clone were community-acquired and were mainly observed in young Bedouin children, causing skin and soft-tissue infections (SSTI). Comparisons between the new clone and other CA-MRSA and HA-MRSA strains are shown in Table 1. All isolates of the pediatric clone were susceptible to TMP/SMX, ciprofloxacin, gentamicin, tetracycline, rifampicin and vancomycin; 17.8% were nonsusceptible to erythromycin and clindamycin (Table 2).ConclusionThe pediatric CA-MRSA clone, previously described only in sporadic cases in Israel, is emerging among previously healthy, young Bedouin children, typically causing SSTI. Isolates are susceptible to a variety of non-β lactam antibiotics.Disclosures All authors: No reported disclosures.

Longitudinal Monitoring of Successive Commercial Layer Flocks forSalmonella entericaSerovar Enteritidis

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

In vivo evolution of tigecycline-non-susceptible Klebsiella pneumoniae strains in patients: relationship between virulence and resistance

Tigecycline resistance among Klebsiella pneumoniae isolates has been increasingly reported. We aimed to investigate the relationship among in vivo acquisition of tigecycline resistance in K. pneumoniae clinical isolates, the underlying molecular mechanisms and bacterial virulence. Clinical isolates of K. pneumoniae from the same patient in a medical centre in Taiwan that were initially tigecycline-susceptible (TS) and then became tigecycline-non-susceptible (TNS) were identified. Clinical data were collected. All isolates were subjected to MIC determination by Etest, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), virulence factor determination, and growth rate and mouse lethality studies. Quantitative RT-PCR was performed to analyse acrA, oqxA, ramA and rarA expressions. The presence of mutations in acrR, ramR, oqxR and rpsJ were analysed by DNA sequencing. Five isogenic paired isolates were determined by PFGE fingerprinting. TNS K. pneumoniae appeared after treatment with a variety of antibiotics among patients infected with TS K. pneumoniae. TNS K. pneumoniae isolates were associated with upregulation of RamA and/or RarA and the corresponding AcrAB and/or OqxAB efflux pump(s), respectively. Various mutations in negative regulatory genes (ramR and oqxR) accounted for overexpression of ramA and rarA, respectively. Three of the five paired isolates showed similar growth rates and virulence between TS and TNS isolates. Two TNS K. pneumoniae strains belonging to capsular types K1 and K20 retained their high virulence. In conclusion, some TNS K. pneumoniae strains derived from TS isolates did not compromise their virulence. Dissemination of these highly pathogenic and resistant strains would be of major concern in the future.

Prevalence and Amounts of Salmonella Found on Raw California Inshell Pistachios

After harvest, pistachios are hulled with mechanical abrasion and then separated in a float tank containing water; the nuts that float (∼15%; floaters) and those that sink (∼85%; sinkers) are dried and stored separately. To determine the prevalence of Salmonella in pistachios, a total of 3,966 samples (1,032 floaters and 2,934 sinkers) were collected within 4 months of the 2010, 2011, and 2012 harvests from storage silos (12 samples from each silo, in most cases) and were stored at 4°C; 100-g subsamples were enriched for the presence of Salmonella. Twenty-one of the floater samples and 11 of the sinker samples were positive for Salmonella: 2.0% prevalence (95% confidence interval [CI], 1.3 to 3.1%) and 0.37% prevalence (95% CI, 0.21 to 0.67%), respectively, for a weighted average prevalence of 0.61%. Levels of Salmonella were determined for positive samples using a most-probable-number (MPN) method with multiple 50-g, three 5.6-g, and three 0.56-g subsamples. Geometric mean levels of Salmonella in floaters and sinkers were 0.66 MPN/100 g (0.14 to 5.3 MPN/100 g) and 0.18 MPN/100 g (0.10 to 0.62 MPN/100 g), respectively. Seven different serovars were identified among the isolates, with nine pulsed-field gel electrophoresis fingerprints; as many as four serovars were isolated from some samples. Salmonella serovars Montevideo (44%), Enteritidis (19%), Senftenberg (16%), Worthington (12%), and Liverpool (9.4%) were most commonly isolated from the initial 100-g samples. The prevalence and levels of Salmonella in pistachios are within those observed for other tree nuts, but the limited number of serovars isolated suggests a narrow and persistent contamination source.

Genotypic, size and morphological diversity of virioplankton in a deep oligomesotrophic freshwater lake (Lac Pavin, France)

We examined changes in morphological and genomic diversities of viruses by means of transmission electronic microscopy and pulsed field gel electrophoresis (PFGE) over a nine-month period (April–December 2005) at four different depths in the oligomesotrophic Lac Pavin. We found that the majority of viruses in this lake belonged to the family of Siphoviridae or were untailed, with capsid sizes ranging from 30 to 60nm, and exhibited genome sizes ranging from 15 to 45kb. On average, 12 different genotypes dominated each of the PFGE fingerprints. The highest genomic viral richness was recorded in summer (mean=14 bands per PFGE fingerprint) and in the epilimnion (mean=13 bands per PFGE fingerprint). Among the physico-chemical and biological variables considered, the availability of the hosts appeared to be the main factor regulating the variations in the viral diversity.

Surveillance on the etiology and molecular epidemiology of Shigella sonnei isolated in Henan province from 2011 to 2014

To detect and analyze the distribution of virulence factors, antimicrobial resistance and pulsed field gel electrophoresis (PFGE) patterns of Shigella sonnei, isolated in Henan province from 2011 to 2014. Samples of diarrhea patients were collected and isolated with SS selective culture medium in 37 ℃ for 18-24 hours. All strains were identified under the Kligler iron agar/motility-indol-urea biochemical action and API20E biochemical system. Serological typing and prepared DNA template were carried out with thermal cracking method and multiplex PCR, to detect the virulence genes of Shigella sonnei. According to the molecular typing method and K-B drug susceptibility testing method published by the international PulseNet bacterial infectious disease monitoring network and USA clinical and Laboratory Standards Institute, antimicrobial susceptibility tests and PFGE molecular characteristics of these positive strains isolated from sentinel hospitals patients stool samples were analyzed. Among the 98 strains of Sonnei type Ⅰ and 118 strains of Sonnei type Ⅱ, all the strains carried carry different virulence genes including SHET-1B, SHET-2, ial, ipaH genes, with 4 kinds of virulence gene combination types. All the 216 strains of Shigella sonnei belonged to the multi-drug resistant strains, including 34 isolates resistant to 2-4 kinds of antibiotics(15.7%), 147 isolates to 5-8 kinds of antibiotics (68.1%), 24 to 9-10 kinds of antibiotics (11.1%), 7 to 11 kinds of antibiotics (3.2%), and 4 to 13 kinds of antibiotics (1.9%). A total of 100 strains of Shigella sonnei were divided into 31 molecular patterns, digested by XbaⅠ and PFGE. Each pattern contained 1-13 strains with similarities ranged from 68.6%-100.0%. All the Shigella sonnei strains carried virulence pathogenic factors, presenting serious status on drug resistance. PFGE fingerprinting patterns showed high polymorphism and dominant characteristics. PFGE patterns of partial strains and corresponding antidrug spectrum presented certain relevance and with same aggregation relationship.

English

Hygiene conditions during milking are essential in dairy cattle to ensure the highest level of milk production. The aim of this work was to assess the efficacy of hygiene measures during the manual milking process with no pre- or post-milking antisepsis measure at the Sahelian experimental station of Toukounous (Niger), on the prevalence of California Mastitis Test (CMT)-positive cows, on the identity of bacterial pathogens recovered from positive milk samples and on the clonality of the Staphylococcus aureus isolated. The application of mammary gland washing prior to milking, of teat dipping with chlorhexidine after milking and of milkers’ hand washing and disinfection with sodium hypochlorite significantly decreased the number of CMT-positive cows. However, the percentages of bacteriologically positive milk samples and the relative isolation rates of S. aureus and coagulase-negative staphylococci did not significantly differ according to the presence/absence of antisepsis measure. As for the clonality of the S. aureus isolates, the 53 S. aureus isolated in the absence of hygiene measures belonged to 10 pulsed field gel electrophoresis (PFGE) fingerprints, while, in comparison, only five PFGE fingerprints were identified amongst the 11 S. aureus isolated in the presence of hygiene measures. As a conclusion, the strict application of hygiene and disinfection during hand milking at Toukounous is a major contributor to the decrease of the percentage of CMT-positive cows and, therefore, of mastitis prevalence and incidence. The reasons for the persistence of the remaining S. aureus after application of hygiene measures may be numerous; one of them could be that they belong to more resistant clones. Key words: Mastitis, antisepsis, manual milking, Azawak zebu, Niger.

Molecular epidemiology analysis of Salmonella enterica serotype Typhi used Pulsed-Field Gel Electrophoresis (PFGE)

Strain typing is an integral part of molecular epidemiology used to discern the clonality of Salmonella Typhi involved in local epidemics. The purpose of this study is to identify sporadic Salmonella enterica serotype Typhi by conventional and molecular methods that include characterization by Pulsed Field Gel Electrophoresis (PFGE) and to present molecular epidemiology analysis. Thirty isolstes of Salmonella typhi from sporadic clinical cases of typhoid fever were obtained. They represent cases from Baghdad, Basra, Babylon and Diala provinces during the period between June 2005 to July 2006. Two biotypes were obtained, 26 isolates under biotype I and four under biotype II. Two antibiogram patterns were obtained: twenty-nine isolates were susceptible to all antibiotic used while the remaining isolate was of different pattern. Plasmid profiling allowed little or no differentiation amongst these isolates. Only 4 (13.3%) isolates were found to contain plasmids which were of three patterns, the majority of strains 26 (86.7%) isolates did not show any plasmid. BOX-PCR fingerprinting has revealed 9 distinct patterns.Cluster analysis by UPGMA based dendrogram revealed six clusters with 90% similarity. Pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNAs from these Salmonella Typhi isolates was performed using the restriction endonucleases XbaI (5'-TCTAGA-3') and SpeI (5'-ACTAGT-3'). XbaI-based analysis was superior to SpeI restriction patterns. PFGE fingerprinting with XbaI restriction have yielded sixteen distinct patterns. Cluster analysis by UPGMA based dendrogram revealed seven clusters with 90% similarity. PFGE fingerprinting with the comparative use of the XbaI and SpeI endonucleases have proved high discriminatory value to other molecular methods and a helpful tool for the epidemiological typing of Salmonella Typhi isolates.