Frequency and diversity of carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA.

As safe agents of last resort, carbapenems are reserved for the treatment of infections caused by multidrug-resistant organisms. The impact of ß-lactam antibiotics, cefotaxime, and meropenem on the frequency and diversity of carbapenemase-producing organisms recovered from environmental samples has not been fully established. Therefore, this methodological study aimed at determining ß-lactam drugs used in selective enrichment and their impact on the recovery of carbapenemase-producing Enterobacterales (CPE) from untreated wastewater. We used a longitudinal study design where 1L wastewater samples were collected weekly from wastewater treatment plant (WWTP) influent and quarterly from contributing sanitary sewers in Columbus, Ohio USA with 52 total samples collected. Aliquots of 500 mL were passed through membrane filters of decreasing pore sizes to enable all the water to pass through and capture bacteria. For each sample, the resulting filters were placed into two modified MacConkey (MAC) broths, one supplemented with 0.5 µg/mL of meropenem and 70 µg/mL of ZnSO4 and the other supplemented with 2 µg/mL cefotaxime. The inoculated broth was then incubated at 37° C overnight, after which they were streaked onto two types of correspondingly-modified MAC agar plates supplemented with 0.5 µg/mL and 1.0 µg/mL of meropenem and 70 µg/mL of ZnSO4 and incubated at 37°C overnight. The isolates were identified based on morphological and biochemical characteristics. Then, up to four distinct colonies of each isolate's pure culture per sample were tested for carbapenemase production using the Carba-NP test. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) MALDI-TOF MS was used to identify carbapenemase-producing organisms. In total 391 Carba-NP positive isolates were recovered from the 52 wastewater samples: 305 (78%) isolates had blaKPC, 73 (19%) carried blaNDM, and 14 (4%) harbored both blaKPC and blaNDM resistance genes. CPE genes of both blaKPC and blaNDM were recovered in both types of modified MAC broths, with 84 (21%) having a blaKPC gene, 22 (6%) carrying blaNDM and 9 (2%) harbored both a blaKPC and blaNDM of isolates recovered from MAC medium incorporated with 0.5ug/mL meropenem and 70ug/mL ZnSO4. The most prevalent isolates were Klebsiella pneumoniae, Escherichia coli, and Citrobacter spp.

Nontyphoidal Salmonella Invasive Disease: Challenges and Solutions.

Nontyphoidal Salmonella are a leading cause of community-onset bacteremia and other serious infections in sub-Saharan African countries where large studies indicate that they are an uncommon cause of moderate-to-severe diarrhea. Approximately 535 000 nontyphoidal Salmonella invasive disease illnesses and 77 500 deaths were estimated to occur in 2017; 422 000 (78.9%) illnesses and 66 500 (85.9%) deaths in countries in sub-Saharan Africa. Lineages of Salmonella enterica serovar Typhimurium sequence type (ST) 313 and lineages of Salmonella enterica serovar Enteritidis ST11 dominate as causes of invasive disease. A major reservoir for these specific strains outside of humans has not been identified to date. Human fecal shedding of such strains is common in areas where nontyphoidal Salmonella invasive disease incidence is high. The case-fatality ratio of nontyphoidal Salmonella invasive disease is approximately 15%. Early diagnosis and treatment are needed to avert fatal outcomes. Antimicrobial resistance, including multiple drug resistance, decreased fluoroquinolone susceptibility, and resistance to third-generation cephalosporins, is increasing in prevalence and is likely to further compromise patient outcomes. Naturally acquired immunity against invasive disease develops in children aged >3 years in endemic areas, likely mediated in part by the sequential acquisition of T-cell immunity, followed by antigen-specific immunoglobulin G antibodies. Vaccines in preclinical or clinical development include live-attenuated S. enterica serovar Typhimurium, nontyphoidal S. enterica core and O-polysaccharide glycoconjugates, multiple antigen-presenting system complexes, and generalized modules for membrane antigens vaccines. The latter are in phase I trials in Europe and Africa. Both vaccine use, and other effective, evidence-based nonvaccine interventions, are needed to prevent and control nontyphoidal Salmonella invasive disease.

Molecular epidemiology of geographically dispersed Vibrio cholerae, Kenya, January 2009-May 2010.

Numerous outbreaks of cholera have occurred in Kenya since 1971. To more fully understand the epidemiology of cholera in Kenya, we analyzed the genetic relationships among 170 Vibrio cholerae O1 isolates at 5 loci containing variable tandem repeats. The isolates were collected during January 2009-May 2010 from various geographic areas throughout the country. The isolates grouped genetically into 5 clonal complexes, each comprising a series of genotypes that differed by an allelic change at a single locus. No obvious correlation between the geographic locations of the isolates and their genotypes was observed. Nevertheless, geographic differentiation of the clonal complexes occurred. Our analyses showed that multiple genetic lineages of V. cholerae were simultaneously infecting persons in Kenya. This finding is consistent with the simultaneous emergence of multiple distinct genetic lineages of V. cholerae from endemic environmental reservoirs rather than recent introduction and spread by travelers.

Molecular characterisation of Vibrio cholerae O1 strains carrying an SXT/R391-like element from cholera outbreaks in Kenya: 1994-2007.

BACKGROUND: Over the last decade, cholera outbreaks in parts of Kenya have become common. Although a number of recent studies describe the epidemiology of cholera in Kenya, there is paucity of information concerning the diversity and occurrence of mobile genetic elements in Vibrio cholerae strains implicated in these outbreaks. A total of 65 Vibrio cholerae O1 El Tor serotype Inaba isolated between 1994 and 2007 from various outbreaks in Kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements (ICEs), conjugative plasmids and for their genotypic relatedness. RESULTS: All the strains were haemolytic on 5% sheep blood and positive for the Vibrio cholerae El Tor-specific haemolysin toxin gene (hylA) by PCR. They all contained strB, sulII, floR and the dfrA1 genes encoding resistance to streptomycin, sulfamethoxazole, chloramphenicol and trimethoprim respectively. These genes, together with an ICE belonging to the SXT/R391 family were transferable to the rifampicin-resistant E. coli C600 en bloc. All the strains were negative for integron class 1, 2 and 3 and for transposase gene of transposon Tn7 but were positive for integron class 4 and the trpM gene of transposon Tn21. No plasmids were isolated from any of the 65 strains. All the strains were also positive for all V. cholera El Tor pathogenic genes except the NAG- specific heat-stable toxin (st) gene. None of the strains were positive for virulence genes associated with the V. cholerae classical biotype. All the strains were positive for El Tor-specific CTXphi bacteriophage rstrR repressor gene (CTXETPhi) but negative for the Classical, Calcutta, and the Environmental repressor types. Pulse Field Gel Electrophoresis (PFGE) showed that regardless of the year of isolation, all the strains bearing the SXT element were clonally related. CONCLUSIONS: This study demonstrates that the V. cholerae O1 strains carrying an SXT/R391-like element implicated in recent cholera outbreaks in Kenya has not changed significantly between 1994 and 2007 and are clonally related.

High incidence of enteroaggregative Escherichia coli among food handlers in three areas of Kenya: a possible transmission route of travelers' diarrhea.

BACKGROUND: Contaminated food and water are acknowledged vehicles for the transmission of travelers' diarrhea (TD). Importance of food handlers as reservoirs of enteroaggregative Escherichia coli (EAEC), enteropathogenic E coli (EPEC), and Shiga toxin-producing E coli (STEC) causing TD has not been clearly demonstrated. METHODS: We undertook a 1-year prospective study to determine the presence and selected risk factors of carriage of EAEC, EPEC, and STEC by 1,399 food handlers working in tourist hotels in three popular tourist destinations of Kenya. Enterotoxigenic E coli (ETEC) was not sought in this study. RESULTS: During the period April 2003 to May 2004, EAEC harboring the aggR gene were detected from 29 (2.1%) subjects and EPEC harboring the eaeA gene and STEC harboring the stx2 gene were detected from 11 (0.8%) and 2 (0.1%) of the study subjects, respectively. Mean age of subjects with EAEC was significantly lower (24.6 y) than the rest of the study population (28.2 y) (p < 0.05). Pit latrines usage was significantly associated with the isolation of EAEC (<0.001) but not with EPEC and STEC. Four of the 29 EAEC isolates were sensitive to all antibiotics tested, and 19 (65.5%) were multidrug resistant (MDR). Antibiotic resistance varied from 6.9% for cefuroxime to 72.4% for co-trimoxazole. Six EPEC isolates (6/13, 46.2%) showed multidrug resistance. Cluster analysis of the pulsed-field gel electrophoresis (PFGE) profiles showed that the EAEC isolates belonged to two clonally unrelated genotypes. CONCLUSIONS: We conclude that food handlers working in tourist hotels are important carriers of EAEC that could cause TD and a high proportion of the EAEC are MDR. The isolation of MDR EAEC from food handlers working in tourist hotels is of potential public health importance. There is a need for a study employing molecular methods including PFGE to examine carriage of similar pathogens in food handlers, processed foods, and travelers consuming the food who develop diarrhea.

Phenotypic and genetic characterization of Vibrio cholerae O1 isolated from various regions of Kenya between 2007 and 2010.

INTRODUCTION: Cholera, a disease caused by Vibrio cholerae O1 and O139 remains an important public health problem globally. In the last decade, Kenya has experienced a steady increase of cholera cases. In 2009 alone, 11,769 cases were reported to the Ministry of Public Health and Sanitation. This study sought to describe the phenotypic characteristics of the isolated V. cholerae isolates. METHODS: This was a laboratory based cross-sectional study that involved isolates from different cholera outbreaks. Seventy six Vibrio cholerae O1 strains from different geographical areas were used to represent 2007 to 2010 cholera epidemics in Kenya, and were characterized by serotyping, biotyping, polymerase chain r(PCR), pulsed-field gel electrophoresis (PFGE) and ribotyping along with antimicrobial susceptibility testing. RESULTS: Seventy six Vibrio cholerae O1 strains from different geographical areas were used to represent 2007 to 2010 cholera epidemics in Kenya. Serotype Inaba was dominant (88.2%) compared to Ogawa. The isolates showed varying levels of antibiotic resistance ranging from 100% susceptible to tetracycline, doxycycline, ofloxacin, azithromycin, norfloxacin and ceftriaxone to 100% resistant to furazolidone, trimethoprim-sulfamethoxazole, polymyxin-B and streptomycin. The isolates were positive for ctxA, tcpA (El Tor), rtxC genes and were biotype El Tor variant harboring classical ctxB gene. All the isolates were classified as cholera toxin (CT) genotype 1 as they had mutation in the ctxB at positions 39 and 68. All the isolates had genetically similar NotI PFGE and BglI ribotype patterns. The absence of any observed variation is consistent with a clonal origin for all of the isolates. CONCLUSION: Kenya experienced cholera numerous outbreak from 2007-2010. The clinical Vibrio cholerae O1 isolates from the recent cholera epidemic were serotypes Inaba and Ogawa, Inaba being the predominant serotype. The Vibrio cholerae O1 strains were biotype El Tor variants that produce cholera toxin B (ctx B) of the classical type and were positive for ctxA, tcpA El Tor and rtxC genes.